pCP20大肠敲除质粒

基本信息

复制子:	pSC101 ori
质粒分类:	大肠系列质粒；大肠编辑质粒；大肠Red质粒
质粒大小:	9332bp
克隆菌株:	DH5α等大肠杆菌
培养条件:	30℃，LB，有氧
5'测序引物:	FLP-R（GAGCTTTGTTGTAGGTGGACC）
3'测序引物:	FLP-F（TTGATGCGCTGGCAGTGTTC）

质粒简介

        pCP20是一种氨苄青霉素和CmR质粒，显示了FLP合成的温度敏感复制和热诱导。 用pCP20转化CmR和KmR突变体，在30℃下选择氨苄青霉素抗性转化体，之后在43℃选择性地纯化一些，然后测试所有抗生素抗性的损失。 大多数同时失去了FRT侧翼抗性基因和FLP辅助质粒。

质粒图谱

质粒序列

LOCUS       Exported                9332 bp ds-DNA     circular SYN 27-JUN-2017
DEFINITION  synthetic circular DNA
KEYWORDS    pCP20
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9332)
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, June 27, 2017 from Plasmid
FEATURES             Location/Qualifiers
     source          1..9332
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(561..1220)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1221..1323)
                     /note="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      1875..2097
                     /note="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2145..3095
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of a protein needed
                     for replication with the pSC101 origin"
                     /note="Rep101(Ts)"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     CDS             complement(4557..5417)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5418..5522)
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             complement(6556..7827)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /note="FLP"
                     /note="FLP is a site-specific recombinase from
                     Saccharomyces cerevisiae. Recombination occurs at FRT
                     sequences."
                     /translation="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     CDS             7928..8641
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /note="lambda repressor (ts)"
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
ORIGIN
        1 gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagatca
       61 cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact
      121 ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg ctggatgatg
      181 gggcgattca ggcctggtat gagtcagcaa caccttcttc acgaggcaga cctcagcgcc
      241 acaggtgcgg ttgctggcgc taaccgtttt tatcaggctc tgggaggcag aataaatgat
      301 catatcgtca attattacct ccacggggag agcctgagca aactggcctc aggcatttga
      361 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
      421 agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt
      481 ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt ttaagggcac
      541 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
      601 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
      661 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg
      721 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg
      781 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg
      841 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca
      901 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
      961 ctatcccata tcaccagctc accgtctttc attgccatac ggaattccgg atgagcattc
     1021 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg
     1081 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
     1141 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
     1201 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa
     1261 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga
     1321 tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca
     1381 ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg
     1441 tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag gtgctccagt
     1501 ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg tgcgtaacgg
     1561 caaaagcacc gccggacatc agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     1621 ccgggggact gttgggcgcc tgtagtgcca tttaccccca ttcactgcca gagccgtgag
     1681 cgcagcgaac tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg tcggagacaa
     1741 aaggaatatt cagcgatttg cccgagcttg cgagggtgct acttaagcct ttagggtttt
     1801 aaggtctgtt ttgtagagga gcaaacagcg tttgcgacat ccttttgtaa tactgcggaa
     1861 ctgactaaag tagtgagtta tacacagggc tgggatctat tctttttatc tttttttatt
     1921 ctttctttat tctataaatt ataaccactt gaatataaac aaaaaaaaca cacaaaggtc
     1981 tagcggaatt tacagagggt ctagcagaat ttacaagttt tccagcaaag gtctagcaga
     2041 atttacagat acccacaact caaaggaaaa ggactagtaa ttatcattga ctagcccatc
     2101 tcaattggta tagtgattaa aatcacctag accaattgag atgtatgtct gaattagttg
     2161 ttttcaaagc aaatgaacta gcgattagtc gctatgactt aacggagcat gaaaccaagc
     2221 taattttatg ctgtgtggca ctactcaacc ccacgattga aaaccctaca aggaaagaac
     2281 ggacggtatc gttcacttat aaccaatacg ttcagatgat gaacatcagt agggaaaatg
     2341 cttatggtgt attagctaaa gcaaccagag agctgatgac gagaactgtg gaaatcagga
     2401 atcctttggt taaaggcttt gagattttcc agtggacaaa ctatgccaag ttctcaagcg
     2461 aaaaattaga attagttttt agtgaagaga tattgcctta tcttttccag ttaaaaaaat
     2521 tcataaaata taatctggaa catgttaagt cttttgaaaa caaatactct atgaggattt
     2581 atgagtggtt attaaaagaa ctaacacaaa agaaaactca caaggcaaat atagagatta
     2641 gccttgatga atttaagttc atgttaatgc ttgaaaataa ctaccatgag tttaaaaggc
     2701 ttaaccaatg ggttttgaaa ccaataagta aagatttaaa cacttacagc aatatgaaat
     2761 tggtggttga taagcgaggc cgcccgactg atacgttgat tttccaagtt gaactagata
     2821 gacaaatgga tctcgtaacc gaacttgaga acaaccagat aaaaatgaat ggtgacaaaa
     2881 taccaacaac cattacatca gattcctacc tacataacgg actaagaaaa acactacacg
     2941 atgctttaac tgcaaaaatt cagctcacca gttttgaggc aaaatttttg agtgacatgc
     3001 aaagtaagta tgatctcaat ggttcgttct catggctcac gcaaaaacaa cgaaccacac
     3061 tagagaacat actggctaaa tacggaagga tctgaggttc ttatggctct tgtatctatc
     3121 agtgaagcat caagactaac aaacaaaagt agaacaactg ttcaccgtta catatcaaag
     3181 ggaaaactgt ccatatgcac agatgaaaac ggtgtaaaaa agatagatac atcagagctt
     3241 ttacgagttt ttggtgcatt taaagctgtt caccatgaac agatcgacaa tgtaacagat
     3301 gaacagcatg taacacctaa tagaacaggt gaaaccagta aaacaaagca actagaacat
     3361 gaaattgaac acctgagaca acttgttaca gctcaacagt cacacataga cagcctgaaa
     3421 caggcgatgc tgcttatcga atcaaagctg ccgacaacac gggagccagt gacgcctccc
     3481 gtggggaaaa aatcatggca attctggaag aaatagcgcc tgtttcgttt caggcaggtt
     3541 atcagggagt gtcagcgtcc tgcggttctc cggggcgttc gggtcatgca gcccgtaatg
     3601 gtgatttacc agcgtctgcc aggcatcaat tctaggcctg tctgcgcggt cgtagtacgg
     3661 ctggaggcgt tttccggtct gtagctccat gttcggaatg acaaaattca gctcaagccg
     3721 tcccttgtcc tggtgctcca cccacaggat gctgtactga tttttttcga gaccgggcat
     3781 cagtacacgc tcaaagctcg ccatcacttt ttcacgtcct cccggcggca gctccttctc
     3841 cgcgaacgac agaacaccgg acgtgtattt cttcgcaaat ggcgtggcat cgatgagttc
     3901 ccggacttct tccggattac cctgaagcac cgttgcgcct tcgcggttac gctccctccc
     3961 cagcaggtaa tcaaccggac cactgccacc accttttccc ctggcatgaa atttaactat
     4021 catcccgcgc cccctgttcc ctgacagcca gacgcagccg gcgcagctca tccccgatgg
     4081 ccatcagtgc ggccaccacc tgaacccggt caccggaaga ccactgcccg ctgttcacct
     4141 tacgggctgt ctgattcagg ttatttccga tggcggccag ctgacgcagt aacggcggtg
     4201 ccagtgtcgg cagttttccg gaacgggcaa ccggctcccc caggcagacc cgccgcatcc
     4261 ataccgccag ttgtttaccc tcacagcgtt caagtaaccg ggcatgttca tcatcagtaa
     4321 cccgtattgt gagcatcctc tcgcgtttca tcggtatcat taccccatga acagaaatcc
     4381 cccttacacg gaggcatcag tgactaaacg gggtctgacg ctcagtggaa cgaaaactca
     4441 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4501 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4561 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4621 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4681 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4741 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4801 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4861 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4921 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4981 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5041 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5101 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5161 tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5221 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5281 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5341 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5401 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5461 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5521 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     5581 acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaatttta taaaccgtgg
     5641 agcgggcaat actgagctga tgagcaattt ccgttgcacc agtgcccttc tgatgaagcg
     5701 tcagcacgac gttcctgtcc acggtacgcc tgcggccaaa tttgattcct ttcagctttg
     5761 cttcctgtcg gccctcattc gtgcgctcta ggatcctcta cgccggacgc atcgtggccg
     5821 gcatcaccgg cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg
     5881 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta
     5941 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg
     6001 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg
     6061 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag
     6121 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     6181 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     6241 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     6301 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     6361 tccggtgaga atggcagaat aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac
     6421 gagcgcttcc gaaaatgcaa cgcgagctgc gcacatacag ctcactgttc acgtcgcacc
     6481 tatatctgcg tgttgcctgt atatatatat acatgagaag aacggcatag tgcgtgttta
     6541 tgcttaaatg cgtacttata tgcgtctatt tatgtaggat gaaaggtagt ctagtacctc
     6601 ctgtgatatt atcccattcc atgcggggta tcgtatgctt ccttcagcac taccctttag
     6661 ctgttctata tgctgccact cctcaattgg attagtctca tccttcaatg ctatcatttc
     6721 ctttgatatt ggatcatatg catagtaccg agaaactagt gcgaagtagt gatcaggtat
     6781 tgctgttatc tgatgagtat acgttgtcct ggccacggca gaagcacgct tatcgctcca
     6841 atttcccaca acattagtca actccgttag gcccttcatt gaaagaaatg aggtcatcaa
     6901 atgtcttcca atgtgagatt ttgggccatt ttttatagca aagattgaat aaggcgcatt
     6961 tttcttcaaa gctttattgt acgatctgac taagttatct tttaataatt ggtattcctg
     7021 tttattgctt gaagaattgc cggtcctatt tactcgtttt aggactggtt cagaattcct
     7081 caaaaattca tccaaatata caagtggatc gatcctaccc cttgcgctaa agaagtatat
     7141 gtgcctacta acgcttgtct ttgtctctgt cactaaacac tggattatta ctcccagata
     7201 cttattttgg actaatttaa atgatttcgg atcaacgttc ttaatatcgc tgaatcttcc
     7261 acaattgatg aaagtagcta ggaagaggaa ttggtataaa gtttttgttt ttgtaaatct
     7321 cgaagtatac tcaaacgaat ttagtatttt ctcagtgatc tcccagatgc tttcaccctc
     7381 acttagaagt gctttaagca tttttttact gtggctattt cccttatctg cttcttccga
     7441 tgattcgaac tgtaattgca aactacttac aatatcagtg atatcagatt gatgtttttg
     7501 tccatagtaa ggaataattg taaattccca agcaggaatc aatttcttta atgaggcttc
     7561 cagaattgtt gctttttgcg tcttgtattt aaactggagt gatttattga caatatcgaa
     7621 actcagcgaa ttgcttatga tagtattata gctcatgaat gtggctctct tgattgctgt
     7681 tccgttatgt gtaatcatcc aacataaata ggttagttca gcagcacata atgctatttt
     7741 ctcacctgaa ggtctttcaa acctttccac aaactgacga acaagcacct taggtggtgt
     7801 tttacataat atatcaaatt gtggcataca acctccttag tacatgcaac cattatcacc
     7861 gccagaggta aaatagtcaa cacgcacggt gttagatatt tatcccttgc ggtgatagat
     7921 ttaacgtatg agcacaaaaa agaaaccatt aacacaagag cagcttgagg acgcacgtcg
     7981 ccttaaagca atttatgaaa aaaagaaaaa tgaacttggc ttatcccagg aatctgtcgc
     8041 agacaagatg gggatggggc agtcaggcgt tggtgcttta tttaatggca tcaatgcatt
     8101 aaatgcttat aacgccgcat tgcttacaaa aattctcaaa gttagcgttg aagaatttag
     8161 cccttcaatc gccagagaaa tctacgagat gtatgaagcg gttagtatgc agccgtcact
     8221 tagaagtgag tatgagtacc ctgttttttc tcatgttcag gcagggatgt tctcacctaa
     8281 gcttagaacc tttaccaaag gtgatgcgga gagatgggta agcacaacca aaaaagccag
     8341 tgattctgca ttctggcttg aggttgaagg taattccatg accgcaccaa caggctccaa
     8401 gccaagcttt cctgacggaa tgttaattct cgttgaccct gagcaggctg ttgagccagg
     8461 tgatttctgc atagccagac ttgggggtga tgagtttacc ttcaagaaac tgatcaggga
     8521 tagcggtcag gtgtttttac aaccactaaa cccacagtac ccaatgatcc catgcaatga
     8581 gagttgttcc gttgtgggga aagttatcgc tagtcagtgg cctgaagaga cgtttggctg
     8641 atcggcaagg tgttctggtc ggcgcatagc tgataacaat tgagcaagaa tctgcatttc
     8701 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     8761 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     8821 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     8881 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     8941 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     9001 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     9061 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     9121 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     9181 atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat
     9241 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     9301 tttatcttgt gcaatgtaac atcagagatt tt