pCL-Eco

pCL-Eco

载体基本信息

出品公司:	Ybscience
试剂名称:	pCL-Eco
质粒类型:	哺乳动物载体；逆病毒包装；单质粒包装系统
高拷贝/低拷贝:	高拷贝
克隆方法:	限制性内切酶，多克隆位点
启动子:	CMV
载体大小:	12341 bp
5' 测序引物及序列:	CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’
3' 测序引物及序列:	--
载体标签:	无
载体抗性:	Ampicillin
筛选标记:	无
克隆菌株:	DH5α等
宿主细胞（系）:	包装细胞系如293T
备注:	逆病毒包装载体pCL-Eco是单质粒包装系统，快速产生Helper-free、毒力高的逆病毒颗粒。
稳定性:	瞬表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

 

载体简介

 

逆病毒包装载体pCL-Eco的使用方法

293T Growth Medium:

   450 ml DMEM (JRH Biosciences)
   50 ml FBS (JRH Sciences)
   5 ml Glutamine (200 mM in saline, JRH Sciences)
   2.5 ml Gentamycin (10 mg/ml in water, Sigma)

HC11 Growth Medium:

  5 ml L-Glutamine (200 mM in saline, JRH Sciences)
  2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)
  2.5 ml Gentamycin (10 mg/ml in water, Sigma)
  0.5 ml EGF (10 μg/ml in water, Gibco)
  50 ml FBS (JRH Sciences)
  440 ml RPMI (JRH Sciences)


Day 0: Split 293T cells for transfection:

  Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube
  Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes
  Spin at 1000 rpm in clinical centrifuge for 1-2 minutes
  Resuspend cells in 10 ml growth medium
  Count cells
  Plate cells at 1X106 cells per 100 mm dish


Day 2: Transfect 293T cells:

  Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total 
  volume per 100 mm dish
  Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of
  200 μl

  Mix DNA plasmids in polypropelene tubes (Falcon #2063):
     1 μg DNA of interest
     1 μg pCL-Eco
     Add 200 μl diluted FuGene dropwise to each DNA tube

  Incubate RT 15 min
  Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media
 

Day 3: Change medium:

  • Remove viral medium and CAREFULLY feed with 6 ml growth medium
  This step may reduce a cytostatic factor (produced after transfections) that
  can inhibit growth and infection of your target cells.

  Split HC11 cells for infection
  Seed HC11 cells at 300,000 cells per 100 mm dish
  If using coverslips, incubate with FBS for at least 1 hour prior to plating


Day 4: Infect target (HC11) cells:

  • Collect virus-containing media off 293T cells
  • Syringe filter media through .45 μm filter onto target cells
   (Evaporation O/N and loss in filter leaves about 5 ml per plate)
  • Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)
   (Store polybrene in aliquots to decrease freeze/thaw cycles)
  • Wrap plates in parafilm
  • Spin cells in clinical centrifuge 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Remove viral media and replace with fresh growth media


Day 6+: Harvest HC11 cells:

  • Wash cells with HBSS
  • Scrape remaining cells, pellet and flash freeze for protein/RNA analysis

  Stain cells for beta-gal expression:

  Fixing Solution :
    1.35 ml 37% Formaldehyde
    0.2 ml Gluteraldehyde
    1X PBS to 25 mls

  Staining Solution:
    625μl 40 mg/ml X-gal in DMF
    50 μl 1M MgCL2
    750 μl 100 mM Potassium Ferricyanide
    750 μl 100 mM Potassium Ferrocyanide
    1X PBS to 25 mls
    [100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]
    [100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]

  Store at room temperature, protect from light.
    1. Make -gal solution and warm in 37°C water bath to prevent crystallization
    2. Aspirate off media
    3. Wash cells 1X PBS
    4. Fix cells 5 min at RT in fixing solution
    5. Wash 3X PBS
    6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces
      crystallization)
    7. Stain cells with staining solution at 37°C for 1-24 hrs
    8. Store stained cells in 70% EtOH at 4°C
      (EMBO J. 5: pg 3133, 1986)


Note:

   HC11 and MEC primary culture cells are very adherent and can withstand
   the force of spinning the plates at 1800 rpm. We found that HC11 cells died at
   2200 rpm. However, this is cell type-dependent, and spinning speed should be
   optimized accordingly.
   Expect to see -gal staining at the periphery of the plate. During the spin
   infection the media is pushed to the outside. The cells in the center of the plate
   will not be exposed to virus and will not be efficiently infected with retrovirus.

 

载体序列

LOCUS       pCL-Eco Sequencing Result	12341 bp 	DNA	SYN
DEFINITION  pCL-Eco Sequencing Result
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..12341
                     /organism="pCL-Eco Sequencing Result"
                     /mol_type="other DNA"
     rep_origin      267..886
                     /label="pBR322_origin"
     misc_feature    complement(1160..1179)
                     /label="SV40pro_F_primer"
     rep_origin      complement(1164..1241)
                     /label="SV40_origin"
     terminator      complement(1709..1828)
                     /label="SV40_PA_terminator"
     misc_feature    complement(1723..1742)
                     /label="EBV_rev_primer"
     misc_feature    complement(2253..2300)
                     /label="SV40_3_splice"
     misc_feature    complement(2306..2321)
                     /label="SV40_int"
     misc_feature    2540..2562
                     /label="pGEX_3_primer"
     CDS             complement(3033..5030)
                     /label="ORF frame 1"
                     /translation="MARSTLSKPLKNKVNPRGPLIPLILLMLRGVSTASPGSSPHQVY
                     NITWEVTNGDRETVWATSGNHPLWTWWPDLTPDLCMLAHHGPSYWGLEYQSPFSSPPG
                     PPCCSGGSSPGCSRDCEEPLTSLTPRCNTAWNRLKLDQTTHKSNEGFYVCPGPHRPRE
                     SKSCGGPDSFYCAYWGCETTGRAYWKPSSSWDFITVNNNLTSDQAVQVCKDNKWCNPL
                     VIRFTDAGRRVTSWTTGHYWGLRLYVSGQDPGLTFGIRLRYQNLGPRVPIGPNPVLAD
                     QQPLSKPKPVKSPSVTKPPSGTPLSPTQLPPAGTENRLLNLVDGAYQALNLTSPDKTQ
                     ECWLCLVAGPPYYEGVAVLGTYSNHTSAPANCSVASQHKLTLSEVTGQGLCIGAVPKT
                     HQALCNTTQTSSRGSYYLVAPTGTMWACSTGLTPCISTTILNLTTDYCVLVELWPRVT
                     YHSPSYVYGLFERSNRHKREPVSLTLALLLGGLTMGGIAAGIGTGTTALMATQQFQQL
                     QAAVQDDLREVEKSISNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYA
                     DHTGLVRDSMAKLRERLNQRQKLFESTQGWFEGLFNRSPWFTTLISTIMGPLIVLLMI
                     LLFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKPIEYEP*"
     CDS             complement(4970..8242)
                     /label="ORF frame 2"
                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
                     GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
                     AP*"
     misc_feature    complement(5079..5095)
                     /label="pBMN_5_primer"
                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
                     GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
                     AP*"
     CDS             complement(8570..10186)
                     /label="ORF frame 2"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(9770..10177)
                     /label="gag"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(9783..9799)
                     /label="pBABE_5_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(9815..9837)
                     /label="MSCV_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(9815..9837)
                     /label="pLXSN_5_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(10319..11018)
                     /label="5_LTR"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     promoter        complement(10497..11077)
                     /label="CMV_immearly_promoter"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(10524..10544)
                     /label="CMV_fwd_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    complement(10710..10997)
                     /label="CAG_enhancer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    11342..11364
                     /label="pGEX_3_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     promoter        11523..11551
                     /label="AmpR_promoter"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     gene            11593..112
                     /label="Ampicillin"
                     /gene="Ampicillin"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     CDS             11593..112
                     /label="ORF frame 1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
ORIGIN
    1 TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA
   61 ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA
  121 CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT
  181 CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT
  241 CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT
  301 GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC
  361 GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC
  421 AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC
  481 GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC
  541 GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG
  601 AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA
  661 CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA
  721 TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC
  781 CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG
  841 ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT
  901 CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT
  961 GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC GAACGACCGA
 1021 GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCCA ATACGCAAAC CGCCTCTCCC
 1081 CGCGCGTTGG CCGATTCATT AATGCAGCCT CCAAAAAAGC CTCCTCACTA CTTCTGGAAT
 1141 AGCTCAGAGG CCGAGGCGGC CTCGGCCTCT GCATAAATAA AAAAAATTAG TCAGCCATGG
 1201 GGCGGAGAAT GGGCGGAACT GGGCGGAGTT AGGGGCGGGA TGGGCGGAGT AAGACCCGCA
 1261 CCCGTTCCCA AACTGCTTAT TAAAGGGGGT ATGGAGGTGC TGGACCTTGT GACAGGGCCA
 1321 GACAGTGTGA CAGAAATAGA AGCAGTCCTG TTTTGACAAG TTGCCTCTGG AAGCCTCTAC
 1381 AATGCCTCTC TTCTTTTTCT CCAGAGTAAG CGGAGGCCAG GGGCCCCCGG CCTCTGCTTA
 1441 ATACTAAAAA AAACAGCTGT TGTCATAGTA ATGATTGGGT GGAAACATTC TAGGCCTGGG
 1501 TGGAGAGGCT TTTTGCTTCC TCTTGCAAAA CCACACTGCC CTCTGGAGGG CAGTTGCCTA
 1561 GCAACTAATT AAAAGAGGAT GTCGCACGGC CAGCTGCGGT CAGTTAGTCA CTTCCTGCTT
 1621 AACTGACTTG ACATTTTCTA TTTTAAGAGT CGGGAGGAAA ATTACTGTGT TGGAGGCCCT
 1681 CCGCCATCTT CTGAAGCTGA TCCAGACATG ATAAGATACA TTGATGAGTT TGGACAAACC
 1741 ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA TTTGTGATGC TATTGCTTTA
 1801 TTTGTAACCA TTATAAGCTG CAATAAACAA GTTAACAACA ACAATTGCAT TCATTTTATG
 1861 TTTCAGGTTC AGGGGGAGGT GTGGGAGGTT TTTTAAAGCA AGTAAAACCT CTACAAATGT
 1921 GGTATGGCTG ATTATGATCT CTAGTCAAGG CACTATACAT CAAATATTTT TCCATAATTT
 1981 TCTTGTATAG CAGTGCAGCT TTTTCCTTTG TGGTGTAAAT AGCAAAGCAA GCAAGAGTTC
 2041 TATTACTAAA CACAGCATGA CTCAAAAAAC TTAGCAATTC TGAAGGAAAG TCCTTGGGGT
 2101 CTTCTACCTT TCTCTTCTTT TTTGGAGGAG TAGAATGTTG AGAGTCAGCA GTAGCCTCAT
 2161 CATCACTAGA TGGCATTTCT TCTGAGCAAA ACAGGTTTTC CTCATTAAAG GCATTCCACC
 2221 ACTGCTCCCA TTCATCAGTT CCATAGGTTG GAATCTAAAA TACACAAACA ATTAGAATCA
 2281 GTAGTTTAAC ACATTATACA CTTAAAAATT TTATATTTAC CTTAGAGCTT TAAATCTCTG
 2341 TAGGTAGTTT GTCCAATTAT GTCACACCAC AGAAGTAAGG TTCCTTCACA AAGATCCTCT
 2401 AGCTACCTAG CTAGATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA
 2461 CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC
 2521 AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG
 2581 AAACGCGCGA GGCAGCCAGC TTACCTCCCG GTGGTGGGTC GGTGGTCCCT GGGCAGGGGT
 2641 CTCCCGATCC CGGACGAGCC CCCAAATGAA AGACCCCCGC TGACGGGTAG TCAATCACTC
 2701 AGAGGAGACC CTCCCAAGGA ACAGCGAGAC CACAAGTCGG ATGCAACTGC AAGAGGGTTT
 2761 ATTGGATACA CGGGTACCCG GGCGACTCAG TCAATCGGAG GACTGGCGCC CCGAGTGAGG
 2821 GGTTGTGGGC TCTTTTATTG AGCTCGGGGA GCAGAAGCGC GCGAACAGAA GCGAGAAGCG
 2881 AACTGATTGG TTAGTTCAAA TAAGGCACAG GGTCATTTCA GGTCCTTGGG GCACCCTGGA
 2941 AACATCTGAT GGTTCTCTAG CTTGCCAAAC CTACAGGTGG GGTCTTTCAT TCCCCCCTTT
 3001 TTCTGGAGAC TAAATAAAAT CTTTTATTTT ATCTATGGCT CGTACTCTAT AGGCTTCAGC
 3061 TGGTGATATT GTTGAGTCAA AACTAGAGCC TGGACCACTG ATATCCTGTC TTTAACAAAT
 3121 TGGACTAATC GATTAAGAAT GCAGGGTCCG AAGAGCAAAA TCATTAGGAG TACAATGAGG
 3181 GGTCCCATAA TGGTAGATAT CAAGGTGGTA AACCAAGGGG ATCTGTTAAA CAGTCCCTCA
 3241 AACCATCCTT GAGTTGACTC AAACAGTTTC TGTCTCTGAT TAAGCCTCTC TCTCAATTTG
 3301 GCCATGCTGT CTCTCACTAG TCCTGTGTGG TCCGCATAGA AGCAACATTC TTCTTTTAGA
 3361 GCAGCACACA GCCCTCCTTC TTTTAGAAAT AACAAGTCTA GGCCCCTTCG ATTCTGTAGG
 3421 ACAACTTCAG ACAGGGAAGT GAGAGACTTT TCTAGGTTAG AGATTGATTT TTCAACCTCC
 3481 CTGAGATCAT CCTGTACTGC GGCTTGGAGC TGCTGGAATT GCTGAGTGGC CATTAGAGCA
 3541 GTAGTCCCTG TTCCTATTCC AGCGGCAATT CCCCCCATGG TTAGTCCACC CAATAATAGG
 3601 GCCAGGGTTA ACGACACCGG TTCTCTTTTG TGTCGGTTGG ATCTCTCAAA CAGGCCGTAA
 3661 ACATAGCTGG GGGAATGATA GGTGACTCTT GGCCAGAGTT CGACAAGAAC ACAATAATCA
 3721 GTGGTAAGGT TCAGTATGGT GGTGGAGATG CATGGAGTAA GCCCGGTACT ACAAGCCCAC
 3781 ATGGTACCTG TAGGGGCAAC TAGATAATAG GACCCTCGAC TGCTTGTCTG GGTGGTATTA
 3841 CATAGGGCCT GATGTGTTTT GGGAACTGCT CCTATGCAGA GTCCCTGTCC GGTCACTTCG
 3901 GACAGGGTCA ACTTGTGTTG GGAGGCCACG GAGCAGTTGG CTGGAGCAGA GGTATGGTTG
 3961 GAGTAGGTAC CCAGGACGGC AACCCCTTCG TAGTAGGGGG GTCCCGCTAC TAGACACAAC
 4021 CAGCACTCTT GGGTTTTGTC AGGACTGGTG AGGTTGAGGG CTTGGTAGGC TCCGTCTACT
 4081 AAGTTTAGCA GCCTATTTTC CGTTCCCGCC GGTGGAAGTT GGGTAGGGGA GAGAGGAGTC
 4141 CCACTGGGTG GTTTGGTGAC TGAAGGCGAC TTAACAGGTT TGGGCTTGGA GAGTGGCTGT
 4201 TGGTCTGCCA GAACGGGGTT TGGCCCTATT GGGACGCGGG GTCCTAGATT TTGGTATCTG
 4261 AGTCGGATCC CAAATGTAAG CCCTGGATCT TGTCCGGAGA CATACAAACG TAAGCCCCAG
 4321 TAATGTCCTG TGGTCCAGGA AGTAACCCGT CTCCCGGCGT CTGTAAACCG AATAACTAAG
 4381 GGGTTGCACC ACTTATTATC TTTGCATACC TGGACAGCCT GGTCAGAGGT GAGATTGTTG
 4441 TTTACTGTGA TGAAATCCCA TGATGAGGAG GGCTTCCAGT AAGCTCTACC GGTTGTCTCA
 4501 CAGCCCCAAT AGGCACAGTA GAAGGAGTCT GGACCCCCAC ATGACTTGGA TTCTCGGGGG
 4561 CGGTGGGGCC CGGGGCAAAC ATAAAATCCC TCATTTGATT TATGAGTTGT CTGGTCTAGC
 4621 TTGAGTCTGT TCCAGGCAGT GTTGCACCGA GGGGTGAGGG AGGTTAAAGG TTCTTCGCAG
 4681 TCTCTGGAAC AGCCTGGGCT GCTGCCCCCT GAGCAACAAG GGGGCCCCGG GGGAGAAGAA
 4741 AAAGGGGATT GATATTCTAG CCCCCAATAA GATGGTCCAT GGTGGGCTAA CATACATAAA
 4801 TCTGGGGTAA GGTCAGGCCA CCAGGTCCAC AGAGGGTGGT TGCCAGAAGT TGCCCATACC
 4861 GTCTCCCGAT CTCCATTGGT TACCTCCCAG GTGATATTAT AGACTTGATG AGGACTGGAG
 4921 CCGGGCGAAG CAGTACTGAC CCCTCTGAGC ATCAGAAGAA TTAAGGGGAT TAGGGGGCCT
 4981 CGCGGGTTAA CCTTATTTTT AAGGGGTTTT GAGAGCGTTG AACGCGCCAT GTCAGTCTAG
 5041 AGGATGGTCC ACCCCCGGGG TCGGCAGCCT TCACGTGGGC GGCGTGTATC CAAGCTGCGA
 5101 TGCCGTCTAC TTTGAGGGCG GTGGGGGTGG TCAGCAGGAC TGTGTAAGGT CCTTTCCAGC
 5161 GAGGTTCTAG GTTCTTAGTC TGGTGTCGGC GGACCCACAC TGTGTCGCCG ACTCGGTAAG
 5221 GGTGAGGTAC CACCGGTCGG TCCAGTTGTT CTTGGTAGGC TGCCGCCAGA GGTCTCCAGA
 5281 CTTCGTGCTG GACTAAGTAG AGAGCCTGTA AGTGAGCTTG GAGAGAGGGG CTGTTAGTAA
 5341 CTCTTGTCAT GTCAGGGTCA GGGAAGTTTA CAAGGGGCGG GGGTGCCCCA TATAAGATCT
 5401 CATATGGGGT GAGGCCATGG GGGCCCGGCG TGTTGCGGGC TCGGTACAGG GCTAAGGGGA
 5461 GTAGGAGCAC CCAGTCTCTA GAGCCAGTTG CAAGCGTTAA TTTAGTTAAA GTCTCCTTGA
 5521 TGGTTCTATT CATTCTTTCT ACCTGGCCTG AGCTTTGGGG TCTGTATGCA CAATGTAATT
 5581 TCCAATCAAT CCCCAACAGA TCGGCCACTG TCTGACTCAC CTTGGAGACG AAGGCAGGCC
 5641 CATTGTCAGT TCCCAATACC TGAGGCATGC CGAACCTGGG GAAGATCTCC TCTAGTAGCT
 5701 TCTTGGTTAC GACCTTGGCG GTTTCTTTCT TGGTTGGGAA GGCTTCTATC CAGCCAGAAA
 5761 AGGTATCTAT AAAAACTAGA AGATATTTAT AGCCATACAA TCCGGGCTTT ATCTCGGTGA
 5821 AATCGATCTC CCAATGAGTG CCGGGCCGAT GCCCGCGGAC CCTAGTTCCC TGTTTAACGG
 5881 CAGACTTGCT GGCGTTGACT TGTGCACAAG CTTTGCAGGT CTCAGTGATA TTTTTGAGTG
 5941 TTCGATCCCG GTTCAGCATG TAGTAGGGAC TGTGGCTTCT CTCTAGGAGA GCCTTCATTT
 6001 TTGAGAAGCT GAGGTGAGTC AGCTGATGAA GAAAGTCTAA TAATTCAAAA GTAAACTGGT
 6061 CAGGCATCAC AGGTTTTCCT TGGTAGACCC AATACTTCTT TGTTTTATCA TAAATGGCCC
 6121 CCAACTTGGT TAGGTCCTTT ATATCAGTCA CTGTGTAATG AAAATGTTCT GAGGTGTAGG
 6181 GTGATGAATT TTCTATGAGG AGGGTAGAGG TGTCTGGAGT CTCTGTGATG GCTGCCTTTC
 6241 GGGCCGCTTG GTCAGCCATC CGGTTGCCTC TAGCCTCGGC GCTGTGTCCC TTTTGATGTC
 6301 CTGGACAATG GATTATGCTA AGTCTTTTGG GCAGAAAGAG GGCTTTTAGT AGGGCCAAGA
 6361 TCTCGTCTTT ATTTTTGATC TCTTTGCCTT CTGATGTGAG CAACCCACGC CTTCTGTATA
 6421 TTTCTCCATG GATATGGGCA GTAGCAAAAG CATAACGGCT ATCAGTATAA ACATTTAGCT
 6481 TCTTACCTTC TGCCATCTTT AGGGCCTGGG TGAGTGCTAT CAGTTCAGCC CGCTGAGCGG
 6541 ATGTCCCGGC TGGCAGGGCT TTAGCCCAGA TTACCTCGGT CTCGGTGGTC ACCGCAGCTC
 6601 CCGCCTTACG CTGTCCCTCT TGTAAGAGAC TGCTTCCATC CGTGTACCAG GTGTGGTCGG
 6661 CGTCTGGGAG CGGCTGGTCC GTTAGGTCGG GTCGGGTTCC GTGGGCTTCG GCCAGGATAT
 6721 CAAGGCAGTT GTGTTGCAGC CCTTCCTCAG GCAGTGGGAG CAGCGTAGCC GGGTTCAGGG
 6781 CTACCACCGG TCCGAACTGG ACCCGGTCCG TGTCCAAAAG CAAGGCCTGA TAGTGAGTCA
 6841 TCCGGGCGTT GGAAAGCCAG CGGTCGGGGG GTTGTTTGAC TAGTGCCTCT ACTGCATGGG
 6901 GGGCCAGAAT GACTAGTGGC TGTCCCATGG TTAGCTTGCC TGCATCCTTT GTCAGTACGG
 6961 CAATGGCTGC TACCATCCGT AGGCAAGGGG GCCACCCAGC TGCTACTGGG TCTAGCTTTT
 7021 TGGACAGGTA GGCCACCGGC CGACGCCAAG GTCCCAGTTT TTGCGTTAGG ACACCTTTGG
 7081 CGTAGCCCTG CTTCTCGTCG ACAAAGAGTT CAAAGGGCTT AGTCAAATCT GGCAACCCCA
 7141 GGGCTGGGGC AGTTAGAAGA GCTTGCTTGA TTTCTTGATA GGCCTTTTGT TGGTCTGGGC
 7201 CCCAATTAAA CAGAGTCCCC GTTTTGGTGA GAGGGTACAA GGGGGCTGCC ATTTCTGCAA
 7261 ACCCAGGGAT CCAGAGGCGA CAGAAGCCTG CCGTCCCTAG GAACTCCCTT AGTTGTCGAG
 7321 GGGTCTTCGG AGTAGGCTGC CCCATCACAG TCTCTTTTCT GGCCTCAGTC AGCCATCTCT
 7381 GACCCTCTTT TAGAAGATAC CCCAGATACT TGACCTGTTT CTGGCAAATT TGGGCTTTCT
 7441 TGGCCGAGGC CCGATACCCG AGGTTCCCTA GGGTTTGTAA CAGGGCCCGA GTACCTTGTT
 7501 GGCAGTCTAG CTCAGAAGTG GCGGCCAGCA GTAAGTCATC CACGTACTGT AGCAGGATCA
 7561 AGTCTGGGTG CTGGATCCGG AAGTCTGCTA GGTCTCTGTG CAGTGCCTCA TCAAACAGGG
 7621 TGGGACTGTT TTTGAAACCC TGTGGGAGTC TGGTCCAGGT CAATTGTCCT GAGATTCCCA
 7681 TCTCTGGATC TCTCCACTCA AAGGCGAAGA GAGGCTGACT GGTGGGGTGG AGTCTCAGGC
 7741 AGAAAAAGGC ATCCTTTAAA TCAAGCACAG TGTACCACTG GTGGGACGGT GGGAGCCCGC
 7801 TCAAGAGGTT GTAAGGGTTG GGCACGGTGG GGTGGATGTC TTCCACCCGC TTGTTGACTT
 7861 CTCTCAGATC CTGGACAGGC CTATAATCAT TAGTCCCTGG TTTCTTAACG GGTAGCAGGG
 7921 GCGTGTTCCA GGGGGACTGG CAGGGTACCA GTATTCCCTG GTCCAACAGT CTCTGTATGT
 7981 GGGGCTTGAT CCCCAGTCTG GCTTCTTGTG ACATGGGGTA TTGTTTTATG GACACGGGGG
 8041 TAGAGGTTGC TTTCAGAGGT ATGATCAGAG GAGCTTGGCG AACTGCCAGT CCCATGCCCC
 8101 CGGTTTCCGC CCAGGCCTGA GGAAAATCAG ACAGCCATGT GGACCCTAGA GAAACATCTG
 8161 GCTCTTTTGA GGTCTCATGT AGCCGATACT CATCTTCTAT ATTTAGGGTC AACACTTGCA
 8221 GGGGCTGCCC CATTGGTCCC ATAACCTGAG CTCCTGATCC CTCAAAGTGG ATTTGGGCTT
 8281 TTAGTTTAGT CAGCAAATCT CTTCCTAACA GAGGATAGGG ACAGTCTGGT ACATGGAGGA
 8341 AAGAGTGGGT GACCTTACCG GTAGCTAGAT GTACTTTGCG ATCCGTGGTC CAGCGATACC
 8401 GCTTTCCTCC AGTAGCCCCT TGGACCCAGG CAGACTTATC ACTTAGGGGT CCAGGATTTT
 8461 GGGTCAGCAC GGAGTGTTGG GCCCCAGTAT CTACCAGGAA GGTGACGGGT TGCCCCCCGA
 8521 CTTTGAGGGT TATCCTGGGT TCAGGGGGGG GCTCCTGACC CTGACCTCCC TAGTCATCTA
 8581 GGGTCAGGAG GGAGGTCTGG GGTCTTGGTC CCCGAGGTCC TCGTGGTTTC TTGGGACAAT
 8641 CTTTAGCCCA GTGCCCCTTT TCTTTGCAGT AGGCACACTG GTCGCGATCG AGTTGGGACC
 8701 TCCTTCGTTC TCCTCCCTGT CTATCCTGTT TCTGTCCACT AACGACAGTG GCCAATAGCT
 8761 TGCTCATCTC TCTATGTCTC CTACGATCTC TTTCTTTCTC TTTCTGCTCA TCCTCTGTCC
 8821 TACGGCGTTC TTCTTTTTCC TCTGTTTCTC TCCTGATACG TTCCTCTCTT TCTTCCGGGG
 8881 TTTCTCGTTT ATTAAAGATC TTTTCTGCCT CTCTAACCAA ATCTCCAAGC GTCTTGTTTT
 8941 TTAAATCTTC TAACCTCTCT AACTTTCTCC CAATGTCTGG GGCAGACTGC CAAATGAAAG
 9001 ACATAGACAC ATTAGTTTCT TGCCCTGGGT CCTCAGGGTC ATAAGGAGTG TACCTGCGAT
 9061 AGGCTTCCTT AAGTCTCTCT AGGAAGGCCG AGGGAGACTC ATTGGGCCCT TGTGTTATTC
 9121 CTTTTACCTT GGCCAAATTG GTGGGGCTTC TGCCCGCGTT TTGGAGACCC GCTAGGAGCA
 9181 ACTGGCGATA GTGGACTAGG TGGTTCCTAC CTGCCTGGGT GGTGTAATCC CAGTCTGGGC
 9241 GCTCGAGGGG AAAAGCGGCA TCGACTTCAT TGGGCAGTTG AGTGGGGCGC CCATCATCGC
 9301 CCCGCACCGC CTTTCTAGCC TCTAAGAGCA CCCGTTGTTT TTCTTCTCCG GTCAGCAGAG
 9361 TCCCCAACAG CTGCTGACAG TCGTCCCAGG TGGGCTGATG GGTGATGAGG ACAGACTCGA
 9421 TCAGAGCTGT CAGTTTACCT GGATCTTCAG AAAAAGAAGG GTTATTATTT TTCCAGTTGT
 9481 AAAGGTCAGA AGAGGAGAAC GGCCAGTATT GAAGCTGTCC GTTTCCTCCT GCGCGGAGGG
 9541 GGAATGCCTG CGAGGTAGTG GAGTCGGCCA CAGGGGGCTC CCGTCTCCCA CGTAGGCGAG
 9601 ATGCCATTGG GGAGGGGTCC GGTGCCTCTC CCGCAGGGGT CGCTTCTCCA CCATTTCCGT
 9661 CCCTGTCGGA AGGGGGTGGT CTTGGGTCCC TATAAGGCGG GGGGTCTTCT GTAAGTAGGT
 9721 CGATGAGCGG CCCCCCACTG TCAGAAAGAA CTTGAGGTTT AGGTTTGGCG CCTAGAGAAG
 9781 GAGTGAGGGC TGGATAAAGG GAGGATCGAG GCGGGGTCGA ACGAGGAGGT TCAAGGGGGA
 9841 GAGACGGGGC GGATGGAGGA AGAGGAGGCG GAGGCTTAGG GTGTACAAAG GGCTTGACCC
 9901 AGGGAGGGGG GTCAAAAGCC AAGGCTTCCC AGGTCACGAT GTAGGGGACC TGGTCTGGGT
 9961 GTCCATGCGG GCCAGGTGAA AAGACCTTGA TCTTAACCTG GGTGATGAGG TCTCGGTTAA
10021 AGGTGCCGTC TCGCGGCCAT CCGACGTTAA AGGTTGGCCA TTCTGCAGAG CAGAAGGTAA
10081 CCCAACGTCT CTTCTTGACA TCTACCGACT GGTTGTGAGC GATCCGCTCG ACATCTTTCC
10141 AGTGACCTAA GGTCAAACTT AAGGGAGTGG TAACAGTCTG GCCCATATTT TCAGACAAAT
10201 ACAGAAACAC AGTCAGACAG AGACAACACA GAACGATGCC CAAGCTTGGC CAGCTTACCT
10261 CCCGGTGGTG GGTCGGTGGT CCCTGGGCAG GGGTCTCCCG ATCCCGGACG AGCCCCCAAA
10321 TGAAAGACCC CCGCTGACGG GTAGTCAATC ACTCAGAGGA GACCCTCCCA AGGAACAGCG
10381 AGACCACAAG TCGGATGCAA CTGCAAGAGG GTTTATTGGA TACACGGGTA CCCGGGCGAC
10441 TCAGTCAATC GGAGGACTGG CGCGCCGAGT GAGGGGTTGT GGGCTCTTTT ATTGAGCTCT
10501 GCTTATATAG ACCTCCCACC GTACACGCCT ACCGCCCATT TGCGTCAATG GGGCGGAGTT
10561 GTTACGACAT TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAACTCC CATTGACGTC
10621 AATGGGGTGG AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA
10681 CTGCCAAAAC CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT
10741 AGGAAAGTCC CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT
10801 GACGTCAATA GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT
10861 TTACCGTAAA TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG
10921 AACATACGTC ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC
10981 ATTTACCGTA AGTTATGTAA CGCGGAACTC CATATATGGG CTATGAACTA ATGACCCCGT
11041 AATTGATTAC TATTAATAAC TAGTCAATAA TCAATGTCAA CGCGCTGGCG TAATAGCGAA
11101 GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA ATGGCGCCTG
11161 ATGCGGTATT TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATATG GTGCACTCTC
11221 AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC AACACCCGCT
11281 GACGCGCCCT GACGGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC
11341 TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGACGAAAG
11401 GGCCTCGTGA TACGCCTATT TTTATAGGTT AATGTCATGA TAATAATGGT TTCTTAGACG
11461 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA
11521 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA
11581 AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA
11641 TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT
11701 CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG
11761 AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC
11821 GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT
11881 CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA
11941 GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT
12001 CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT
12061 GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT
12121 GACACCACGA TGCCTGCAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA
12181 CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA
12241 CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT
12301 GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG A