| 质粒名称: | pCNA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 135191 |
| 存储实验室: | Hongqing Fang |
|
载体骨架基本信息 |
|
| 载体名称: | pKOBEG |
| 完整质粒大小: | 7312 bp |
| 载体类型: | Bacterial Expression, Synthetic Biology |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | I-CreI lamba Red |
| 物种来源: | Synthetic |
| 基因突变: | NONE |
| 启动子: | araBAD |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI(未破坏) |
| 3’克隆位点: | HindIII(未破坏) |
| 5’测序引物 | 未知 |
|
文献引用方法 |
|
| 材料和方法部分: | pCNA was a gift from Hongqing Fang (Addgene plasmid # 135191 ; http://n2t.net/addgene:135191 ; RRID:Addgene_135191) |
| 参考文献部分: | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome. Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H. PLoS One. 2017 Oct 24;12(10):e0186891. doi: 10.1371/journal.pone.0186891. eCollection 2017. 10.1371/journal.pone.0186891 PubMed 29065183 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pCNA 7312 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Arabinose inducible expression of lamba-Red recombinase proteins and
I-CreI..
ACCESSION .
VERSION .
KEYWORDS pCNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7312)
AUTHORS Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H
TITLE An electroporation-free method based on Red recombineering for
markerless deletion and genomic replacement in the Escherichia coli
DH1 genome.
JOURNAL PLoS One. 2017 Oct 24;12(10):e0186891. doi:
10.1371/journal.pone.0186891. eCollection 2017.
PUBMED 29065183
REFERENCE 2 (bases 1 to 7312)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7312
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 473..1153
/codon_start=1
/gene="exo"
/product="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
/label=Exo
/note="creates 3' single-stranded DNA tails"
/translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
CDS complement(1651..2601)
/codon_start=1
/gene="rep101 encoding an A56V mutation"
/product="temperature-sensitive version of the RepA protein
needed for replication with the pSC101 origin (Armstrong et
al., 1984)"
/label=Rep101(Ts)
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin 2649..2871
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
primer_bind complement(3464..3482)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 3550..3654
/gene="bla"
/label=AmpR promoter
CDS 3655..4515
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(3873..3892)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
CDS complement(4835..5713)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 5740..6024
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
primer_bind 5947..5966
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
CDS 6581..6997
/codon_start=1
/gene="gam"
/product="inhibitor of the host RecBCD nuclease in the
lambda Red system"
/label=Gam
/note="protects the double-stranded DNA substrate for
recombination"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 7003..476
/codon_start=1
/gene="bet"
/product="single-stranded DNA binding recombinase in the
lambda Red system"
/label=Beta
/note="mediates pairing of a single-stranded DNA with a
complementary target"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
ORIGIN
1 gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg
61 atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc
121 agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct
181 tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat
241 acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg
301 agattaacac tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct
361 gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag
421 taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc
481 ggacattatc ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggaagatgc
541 gtggcacaaa ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa
601 accccgctcc ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc
661 tgaggtttgc accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca
721 gtacgagaac gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc
781 cccgatcatc tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag
841 tgacggcaac ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg
901 gctcggtggt ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg
961 ggtgacgcga aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg
1021 cctgcattat gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt
1081 gccggagttc atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga
1141 gcaatggcga tgacgcatcc tcacgataat aagcttcctg ctgaacatca aaggcaagaa
1201 aacatctgtt gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaaa
1261 acgcaaaaga aaatgccgat attgactacc ggaagcagtg tgaccgtgtg cttctcaaat
1321 gcctgattca ggctgtctat gtgtgactgt tgagctgtaa caagttgtct caggtgttca
1381 atttcatgtt ctagttgctt tgttttactg gtttcacctg ttctattagg tgttacatgc
1441 tgttcatctg ttacattgtc gatctgttca tggtgaacag ctttaaatgc accaaaaact
1501 cgtaaaagct ctgatgtatc tatctttttt acaccgtttt catctgtgca tatggacagt
1561 tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct
1621 tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt
1681 ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt
1741 actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa
1801 agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt
1861 tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat
1921 ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac
1981 caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
2041 gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc
2101 aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca
2161 ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt
2221 tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa
2281 tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
2341 aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc
2401 ataagcattt tccctactga tgttcatcat ctgaacgtat tggttataag tgaacgatac
2461 cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa
2521 aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt
2581 gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
2641 attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg
2701 taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc
2761 cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa
2821 gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta
2881 gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
2941 accttaaaac cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat
3001 tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg
3061 ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga
3121 ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt
3181 ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
3241 ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat
3301 gcacccagta aggcagcggt atcatcaaca ggcttacccg tcttactgtc ggggatcgac
3361 gctctccctt atgcgactcc tgcacctttc gtcttcgaat aaatacctgt gacggaagat
3421 cacttcgcag aataaataaa tcctggtgtc cctggacgaa agggcctcgt gatacgccta
3481 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
3541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
3601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
3661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
3721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
3781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
3841 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
3901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
3961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
4021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
4081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
4141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
4201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
4261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
4321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
4381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
4441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
4501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgcgtaattt
4561 ttttaaggca gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat
4621 aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg
4681 gtcgttaaat agccgcttat gtctattgct ggtctcggta cccggggatc ctctagagtc
4741 gacctgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact
4801 ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat
4861 tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa
4921 atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag
4981 gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc
5041 ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc
5101 aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt
5161 actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt
5221 ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc
5281 cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg
5341 cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat
5401 gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat
5461 gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa
5521 caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat
5581 aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg
5641 gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt
5701 gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt
5761 gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa
5821 ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta
5881 acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac
5941 tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga cgctttttat
6001 cgcaactctc tactgtttct ccatacccgt ttttttgggc tagctaagaa ggagatatac
6061 atatgttata cctggcaggt ttcgtggacg gtgacggtag catcatcgct cagattaaac
6121 caaaccagtc ttataagttt aaacatcagc tgagcttgac ctttcaggtg actcaaaaga
6181 cccagcgccg ttggtttctg gacaaactag tggatgaaat tggcgttggt tacgtacgtg
6241 atcgcggttc cgtttccgat tacatcttaa gcgaaatcaa gccgctgcac aacttcctga
6301 ctcaactgca gccgtttctg aaactgaaac agaaacaggc aaacctggtt ctgaaaatta
6361 tcgaacagct gccgtctgca aaagaatccc cggacaaatt cctggaagtt tgtacctggg
6421 tggatcagat tgcagctctg aacgattcta agactcgtaa aaccacttct gaaaccgttc
6481 gtgctgtgct ggacagcctg agcgagaaga agaaatcctc cccggcggcc gactaatcta
6541 gcgaaaagat gtttcgtgaa gccgtcgacg cttataaaaa atggatatta atactgaaac
6601 tgagatcaag caaaagcatt cactaacccc ctttcctgtt ttcctaatca gcccggcatt
6661 tcgcgggcga tattttcaca gctatttcag gagttcagcc atgaacgctt attacattca
6721 ggatcgtctt gaggctcaga gctgggcgcg tcactaccag cagctcgccc gtgaagagaa
6781 agaggcagaa ctggcagacg acatggaaaa aggcctgccc cagcacctgt ttgaatcgct
6841 atgcatcgat catttgcaac gccacggggc cagcaaaaaa tccattaccc gtgcgtttga
6901 tgacgatgtt gagtttcagg agcgcatggc agaacacatc cggtacatgg ttgaaaccat
6961 tgctcaccac caggttgata ttgattcaga ggtataaaac gaatgagtac tgcactcgca
7021 acgctggctg ggaagctggc tgaacgtgtc ggcatggatt ctgtcgaccc acaggaactg
7081 atcaccactc ttcgccagac ggcatttaaa ggtgatgcca gcgatgcgca gttcatcgca
7141 ttactgatcg ttgccaacca gtacggcctt aatccgtgga cgaaagaaat ttacgccttt
7201 cctgataagc agaatggcat cgttccggtg gtgggcgttg atggctggtc ccgcatcatc
7261 aatgaaaacc agcagtttga tggcatggac tttgagcagg acaatgaatc ct
//
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