基本信息
| 启动子: | T7/Lac |
| 复制子: | ColA ori |
| 终止子: | T7 terminator |
| 质粒分类: | 大肠杆菌载体；双表达框质粒 |
| 质粒大小: | 3719bp |
| 质粒标签: | N-6×His,C-S |
| 原核抗性: | 卡那青霉素Kan |
| 克隆菌株: | DH5α |
| 培养条件: | 37℃，有氧，LB |
| 表达宿主: | BL21(DE3) |
| 培养条件: | 37℃，有氧，LB |
| 诱导方式: | IPTG或乳糖及其类似物 |
| 5'测序引物: | ACYCDuetUP1：GGATCTCGACGCTCTCCCT DuetUP2:TTGTACACGGCCGCATAATC |
| 3'测序引物: | DuetDOWN1:GATTAGCGGCCGTGTACAA T7-ter:TGCTAGTTATTGCTCAGCGG |
| 备注: | 同时表达两个外源蛋白 |
质粒简介
pCOLADuet-1 is designed for the coexpression of two target genes from a single plasmid. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). MCS-1 encodes the six-amino acid His•Tag sequence for the creation of a N-terminal fusion and MCS2 encodes the 15 amino acid S•Tag™ peptide after the last restriction site for the creation of a C-terminal fusion if desired. Genes inserted into MCS-1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer. Genes inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer. The vector has the COLA replicon from ColA(1) and the kanamycin resistance gene. This vector can be transformed into the same cell with plasmids containing compatible origins of replication and drug resistance genes for coexpression of up to 8 target genes.
质粒图谱
质粒序列
LOCUS Exported 3719 bp ds-DNA circular SYN 26-8-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3719)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-8-26 from MLCC
FEATURES Location/Qualifiers
source 1..3719
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 3..27
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
CDS 83..100
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
promoter 214..232
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 233..257
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
CDS 366..410
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/note="S-Tag"
/translation="KETAAAKFERQHMDS"
terminator 462..509
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(739..1554)
/codon_start=1
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
promoter complement(1555..1646)
/gene="bla"
/note="AmpR promoter"
rep_origin complement(1664..2299)
/direction=LEFT
/note="ColA ori"
/note="Plasmids containing the ColA origin of replication
can be propagated in E. coli cells that contain additional
plasmids with compatible origins."
CDS complement(2497..3579)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(3580..3657)
/gene="lacI"
/note="lacI promoter"
/note="
"
promoter 3703..2
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
301 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
361 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
421 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
481 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
541 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
601 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt
661 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
721 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
781 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
841 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
901 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
961 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
1021 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
1081 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca
1141 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
1201 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
1261 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
1321 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
1381 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
1441 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
1501 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt
1561 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
1621 tgaatgtatt tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg
1681 ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct
1741 ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
1801 aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc
1861 cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt
1921 gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg
1981 cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact
2041 ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt
2101 ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag
2161 ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg
2221 gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca agaagatcct
2281 ttacggattc ccgacaccat cactctagat ttcagtgcaa tttatctctt caaatgtagc
2341 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
2401 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
2461 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
2521 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
2581 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
2641 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
2701 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
2761 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
2821 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
2881 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
2941 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
3001 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
3061 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
3121 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
3181 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
3241 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
3301 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
3361 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
3421 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
3481 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
3541 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
3601 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
3661 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
温馨提示：不可用于临床ZL。
