质粒名称: | pComb3XSS |
---|---|
出品公司: | 爱迪基因 |
目录编号: | 63890 |
存储实验室: | Carlos Barbas |
载体骨架基本信息 |
|
载体名称: | pComb3H |
载体抗性: | 氨苄霉素 |
空载体大小: | 3300 bp |
完整质粒大小: | 4900 bp |
载体修饰: | Addition of 6xHis and HA tags for detection and purification, amber stop codon upstream of geneIII to produce soluble protein by switching to a non-suppressor strain of E. coli (e.g. Top10F') |
载体类型: | 细菌表达;phage display |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
|
插入基因名称: | SS Stuffer |
物种来源: | Synthetic |
插入大小: | 1600 bp |
基因又名: | SfiI Stuffer |
启动子 | lacZ |
标签/融合蛋白: |
6x His (C terminal on backbone)
HA tag (C terminal on backbone)
geneIII (C terminal on backbone)
|
克隆方法: | 限制性内切酶 |
5' 克隆位点: | SfiI(未破坏) |
3' 克隆位点: | SfiI (未破坏) |
5' 测序引物 | 5'-AAG ACA GCT ATC GCG ATT GCA G-3' |
3' 测序引物 | 5'-GCC CCC TTA TTA GCG TTT GCC ATC-3' |
文献引用方法 |
|
材料和方法部分: | pComb3XSS was a gift from Carlos Barbas (爱迪基因 plasmid # 63890) |
参考文献部分: | Methods for the generation of chicken monoclonal antibody fragments by phage display. Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF 3rd. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. S0022175900002210 [pii] PubMed 10986398 |
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LOCUS pComb3XSS 4991 bp ds-DNA circular SYN 10-JAN-2018 DEFINITION 3rd generation plasmid for phage display on modified geneIII, contains stuffer fragment. ACCESSION . VERSION . KEYWORDS pComb3XSS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4991) AUTHORS Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF 3rd TITLE Methods for the generation of chicken monoclonal antibody fragments by phage display. JOURNAL J Immunol Methods. 2000 Aug 28;242(1-2):159-81. PUBMED 10986398 COMMENT This file is created by Vector NTI http://www.biofeng.com/ COMMENT ORIGDB|GenBank COMMENT VNTAUTHORNAME|biofeng.com| COMMENT VNTNAME|pComb3XSS| FEATURES Location/Qualifiers source 1..4991 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 109..130 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 145..175 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 183..199 /label=lac operator /bound_moiety="lac repressor encoded by 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)." primer_bind complement(406..422) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(406..422) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(419..441) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 430..446 /label=lac operator /bound_moiety="lac repressor encoded by 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)." promoter complement(454..484) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 499..520 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." CDS complement(574..591) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1584..1907 /codon_start=1 /gene="trxA" /product="E. coli thioredoxin" /label=TrxA /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL DANL" CDS 1962..1979 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1986..2012 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" rep_origin complement(2606..3061) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2743..2764) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2955..2974 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3088..3192 /gene="bla" /label=AmpR promoter CDS 3193..4053 /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(3411..3430) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4224..4812 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4713..4732 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4966..4983 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 181 aattgtgagc ggataacaat tgaattcagg aggaatttaa aatgaaaaag acagctatcg 241 cgattgcagt ggcactggct ggtttcgcta ccgtggccca ggcggccgag ctcgccatgg 301 ctggttgggc agcgagtaat aacaatccag cggctgccgt aggcaatagg tatttcatta 361 tgactgtctc cttggcgact agctagttta gaattcgtaa tcatggtcat agctgtttcc 421 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 481 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 541 cgctttccag tcgggaaacc tgtcgtgtta ctaatgatgg tgatggtgat ggctagtttt 601 gtcacaagat ttgggctcaa ctttcttgtc caccttggtg ttgctgggct tgtgattcac 661 gttgcagatg taggtctggg tgcccaagct gctggagggc acggtcacca cgctgctgag 721 ggagtagagt cctgaggact gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc 781 gcctgagttc cacgacaccg tcgccggttc ggggaagtag tccttgacca ggcagcccag 841 ggccgctgtg cccccagagg tgctcttgga ggagggtgcc agggggaaga ccgatgggcc 901 cttggtggag gctgcggaga cggtgaccgt ggtaccagca gaaacctggc caggctccca 961 ggctcctcat ctatggtaca tccagcaggg ccactggcat cccagacagg ttcagtggca 1021 gtgggtctgg gacagacttc actctcacca tcagcagact ggagcctgaa gattttgcag 1081 tgtactactg tcagcagtat ggtggctcac cgtggttcgg ccaagggacc aaggtggaac 1141 tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga 1201 aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag 1261 tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc 1321 aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact 1381 acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc ttgcccgtca 1441 caaagagctt caacagggga gagtgttagt tctagataat taattaggag gaatttaaaa 1501 tgaaatacct attgcctacg gcagccgctg gattgttatt actcgctgcc caaccagcca 1561 tggccgaggt gcagctgctc gagatgagcg ataaaattat tcacctgact gacgacagtt 1621 ttgacacgga tgtactcaaa gcggacgggg cgatcctcgt cgatttctgg gcagagtggt 1681 gcggtccgtg caaaatgatc gccccgattc tggatgaaat cgctgacgaa tatcagggca 1741 aactgaccgt tgcaaaactg aacatcgatc aaaaccctgg cactgcgccg aaatatggca 1801 tccgtggtat cccgactctg ctgctgttca aaaacggtga agtggcggca accaaagtgg 1861 gtgcactgtc taaaggtcag ttgaaagagt tcctcgacgc taacctggcg tacccgtacg 1921 acgttccgga ctacggttct actagtggcc aggccggcca gcaccatcac catcaccatg 1981 gcgcataccc gtacgacgtt ccggactacg cttcttagga gggtggtggc tctgagggtg 2041 gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2101 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2161 aaaacgtgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2221 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2281 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2341 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2401 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2461 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2521 ttgctaacat actgcgtaat aaggagtctt aagctagcta attaatttaa gcggccgcag 2581 atctgctctc tgaggaggat ctgggaaatt gtaagcgtta atattttgtt aaaattcgcg 2641 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 2701 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 2761 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 2821 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca 2881 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 2941 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta 3001 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 3061 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 3121 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3181 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 3241 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 3301 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 3361 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 3421 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 3481 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 3541 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 3601 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 3661 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 3721 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 3781 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3841 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3901 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3961 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 4021 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 4081 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 4141 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 4201 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 4261 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 4321 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 4381 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 4441 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 4501 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 4561 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 4621 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 4681 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 4741 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 4801 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4861 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4921 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4981 gaggaagcgg a //
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