pAmCyan1-N1

pAmCyan1-N1

载体基本信息

出品公司:	Ybscience
载体名称:	pAmCyan1-N1
质粒类型:	哺乳动物细胞表达载体；荧光报告载体
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 IE
载体大小:	4703 bp
5' 测序引物及序列:	--
3' 测序引物及序列:	--
载体标签:	AmCyan1(C-端）
载体抗性:	卡那霉素
筛选标记:	新霉素（Neomycin）
克隆菌株:	DH5α, HB101
宿主细胞（系）:	常规细胞系，293、CV-1、CHO等
备注:	pAmCyan1-N1载体是青色荧光报告载体，表达C端AmCyan1融合蛋白； AmCyan1是AmCyan的突变体，与AmCyan1相比，编码序列经过了优化，更适合在哺乳动物细胞中的表达； 同时突变了两个氨基酸位点， 改变了发光特点。
稳定性:	瞬表达 或 稳表达
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

 

pAmCyan1-N1载体描述

pAmCyan1-N1 encodes a human codon-optimized variant of wild-type Anemonia majano cyan fluorescent protein, AmCyan1 (1). The AmCyan1 coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for optimal expression in mammalian cells (2). Additionally, an upstream sequence—located just 5' to the AmCyan1 start codon—has been converted to a Kozak consensus translation initiation site(3) to further increase the translation efficiency in eukaryotic cells. Two amino acid substitutions (Asn-34 to Ser; Lys-68 to Met) have been made to enhance the emission characteristics of AmCyan1 (excitation maximum = 458 nm; emission maximum = 489 nm).
The multiple cloning site (MCS) in pAmCyan1-N1 is positioned between the immediate-early promoter of CMV (PCMV IE) and the AmCyan1 coding sequence. Thus, genes cloned into the MCS will be expressed as fusions to the N-terminus of AmCyan1 if they are in the same reading frame as AmCyan1 and there are no intervening stop codons. The SV40 polyadenylation signals (SV40 polyA) downstream of the AmCyan1 gene direct proper processing of the 3'end of AmCyan1 mRNA.

The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express the SV40 T antigen, a pUC origin of replication (pUC ori) for propagation in E. coli, and an f1 origin (f1 ori) for single-stranded DNA production. In addition, a neomycin-resistance cassette— consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (4). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/Kanr gene in E. coli host cells, which can be selected with kanamycin.

Fusions to the N terminus of AmCyan1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAmCyan1-N1 so that it is in frame with the AmCyan1 coding sequence, with no intervening, in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant pAmCyan1-N1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (available from Clontech; Cat. Nos. 631307 & 631308). We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAmCyan1-N1 can also be used simply to express AmCyan1 in a cell line of interest (e.g., as a transfection marker).


Propagation in E. coli
 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue.
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
 E. coli replication origin: pUC
 Copy number: ~500
 Plasmid incompatibility group: pMB1/Col E1

 

载体序列

LOCUS       pAmCyan1-N1	4703 bp 	DNA	SYN
DEFINITION  pAmCyan1-N1
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..4703
                     /organism="pAmCyan1-N1"
                     /mol_type="other DNA"
     promoter        10..562
                     /label="CMV_immearly_promoter"
     misc_feature    65..352
                     /label="CAG_enhancer"
     misc_feature    519..539
                     /label="CMV_fwd_primer"
     promoter        520..589
                     /label="CMV_promoter"
     CDS             679..1368
                     /label="ORF frame 1"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     misc_feature    1582..1601
                     /label="EBV_rev_primer"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     rep_origin      complement(1751..2057)
                     /label="f1_origin"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     promoter        2136..2164
                     /label="AmpR_promoter"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     misc_feature    complement(2230..2250)
                     /label="pBABE_3_primer"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     misc_feature    complement(2236..2451)
                     /label="SV40_enhancer"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     promoter        2248..2516
                     /label="SV40_promoter"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     rep_origin      2415..2492
                     /label="SV40_origin"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     misc_feature    2477..2496
                     /label="SV40pro_F_primer"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS
                     TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE
                     DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK
                     GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH
                     AVAHITSVVPF*"
     CDS             2599..3393
                     /label="ORF frame 1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
     gene            2602..3390
                     /label="NeoR/KanR"
                     /gene="NeoR/KanR"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
     CDS             complement(2908..3444)
                     /label="ORF frame 3"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
     terminator      3568..3837
                     /label="TK_PA_terminator"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
     rep_origin      3985..4604
                     /label="pBR322_origin"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
ORIGIN
    1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG
   61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT
  121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA
  181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC
  241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA
  301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC
  361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG
  421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG
  481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT
  541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA
  601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG
  661 GATCCACCGG TCGCCACCAT GGCCCTGTCC AACAAGTTCA TCGGCGACGA CATGAAGATG
  721 ACCTACCACA TGGACGGCTG CGTGAACGGC CACTACTTCA CCGTGAAGGG CGAGGGCAGC
  781 GGCAAGCCCT ACGAGGGCAC CCAGACCTCC ACCTTCAAGG TGACCATGGC CAACGGCGGC
  841 CCCCTGGCCT TCTCCTTCGA CATCCTGTCC ACCGTGTTCA TGTACGGCAA CCGCTGCTTC
  901 ACCGCCTACC CCACCAGCAT GCCCGACTAC TTCAAGCAGG CCTTCCCCGA CGGCATGTCC
  961 TACGAGAGAA CCTTCACCTA CGAGGACGGC GGCGTGGCCA CCGCCAGCTG GGAGATCAGC
 1021 CTGAAGGGCA ACTGCTTCGA GCACAAGTCC ACCTTCCACG GCGTGAACTT CCCCGCCGAC
 1081 GGCCCCGTGA TGGCCAAGAA GACCACCGGC TGGGACCCCT CCTTCGAGAA GATGACCGTG
 1141 TGCGACGGCA TCTTGAAGGG CGACGTGACC GCCTTCCTGA TGCTGCAGGG CGGCGGCAAC
 1201 TACAGATGCC AGTTCCACAC CTCCTACAAG ACCAAGAAGC CCGTGACCAT GCCCCCCAAC
 1261 CACGTGGTGG AGCACCGCAT CGCCAGAACC GACCTGGACA AGGGCGGCAA CAGCGTGCAG
 1321 CTGACCGAGC ACGCCGTGGC CCACATCACC TCCGTGGTGC CCTTCTGAAG CGGCCGCGAC
 1381 TCTAGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC
 1441 CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA
 1501 TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT
 1561 TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TAAGGCGTAA
 1621 ATTGTAAGCG TTAATATTTT GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT
 1681 TTTAACCAAT AGGCCGAAAT CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA
 1741 GGGTTGAGTG TTGTTCCAGT TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC
 1801 GTCAAAGGGC GAAAAACCGT CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA
 1861 TCAAGTTTTT TGGGGTCGAG GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC
 1921 CGATTTAGAG CTTGACGGGG AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG
 1981 AAAGGAGCGG GCGCTAGGGC GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA
 2041 CCCGCCGCGC TTAATGCGCC GCTACAGGGC GCGTCAGGTG GCACTTTTCG GGGAAATGTG
 2101 CGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA
 2161 CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA
 2221 ACCAGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA
 2281 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT
 2341 CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC
 2401 CCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG
 2461 GCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC
 2521 AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA
 2581 GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT
 2641 TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC
 2701 GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC
 2761 GGTGCCCTGA ATGAACTGCA AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC
 2821 GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG
 2881 GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC
 2941 ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC
 3001 CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT
 3061 CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC
 3121 AAGGCGAGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG
 3181 AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG
 3241 GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC
 3301 GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC
 3361 GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG
 3421 ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA
 3481 GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC
 3541 TCATGCTGGA GTTCTTCGCC CACCCTAGGG GGAGGCTAAC TGAAACACGG AAGGAGACAA
 3601 TACCGGAAGG AACCCGCGCT ATGACGGCAA TAAAAAGACA GAATAAAACG CACGGTGTTG
 3661 GGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC
 3721 CGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA
 3781 GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT GCCATAGCCT
 3841 CAGGTTACTC ATATATACTT TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT
 3901 AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC
 3961 ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC
 4021 GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG
 4081 ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA
 4141 ATACTGTCCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC
 4201 CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT
 4261 GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA
 4321 CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC
 4381 TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC
 4441 CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT
 4501 GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT
 4561 GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC
 4621 TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG
 4681 ATAACCGTAT TACCGCCATG CAT