LOCUS Exported 4130 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of SpyCatcher002 with a peptide tag enabling site-specific biotinylation. ACCESSION . VERSION . KEYWORDS pDEST14-Avitag-SpyCatcher002 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4130) AUTHORS Keeble AH, Banerjee A, Reddington SC, Ferla MP, Howarth M, Khairil Anuar INA TITLE Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics. JOURNAL Angew Chem Int Ed Engl. 2017 Oct 10. doi: 10.1002/anie.201707623. PUBMED 29024296 REFERENCE 2 (bases 1 to 4130) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4130 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 51..73 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 93..110 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 117..161 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 186..206 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 631..648 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(701..719) /label=T7 Term /note="T7 terminator, reverse primer" terminator 715..762 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 799..1254 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(886..905) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1096..1117 /label=F1ori-F /note="F1 origin, forward primer" promoter 1280..1384 /gene="bla" /label=AmpR promoter CDS 1385..2245 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1603..1622) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2416..3004 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2905..2924 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3158..3175 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3190..3329 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3255..3274) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3415..3437 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3431..3622) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac 61 tttaagaagg agatatacat atgtcgtact accatcacca tcaccatcac ggcagcggcc 121 tgaacgatat ttttgaagcg cagaaaattg aatggcatga aggcgattac gacatcccaa 181 cgaccgaaaa cctgtatttt cagggcgcca tggtaaccac cttatcaggt ttatcaggtg 241 agcaaggtcc gtccggtgat atgacaactg aagaagatag tgctacccat attaaattct 301 caaaacgtga tgaggacggc cgtgagttag ctggtgcaac tatggagttg cgtgattcat 361 ctggtaaaac tattagtaca tggatttcag atggacatgt gaaggatttc tacctgtatc 421 caggaaaata tacatttgtc gaaaccgcag caccagacgg ttatgaggta gcaactgcta 481 ttacctttac agttaatgag caaggtcagg ttactgtaaa tggcgaagca actaaaggtg 541 acgctcatac tggatccagt ggtagctaaa tggttgatgc ttgaggatcc gaattcgagc 601 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct 661 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 721 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 781 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 841 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 1021 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 1081 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 1141 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 1201 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg 1261 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 1321 tatgtatccg ctcatgagac aataaccctg gtaaatgctt caataatatt gaaaaaggaa 1381 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 1441 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 1501 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 1561 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 1621 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 1681 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 1741 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 1801 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 1861 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 1921 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 1981 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 2041 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 2101 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 2161 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 2221 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 2281 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 2341 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 2401 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 2461 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 2521 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 2581 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 2641 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 2701 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 2761 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 2821 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 2881 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 2941 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 3001 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 3061 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 3121 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 3181 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa 3241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc 3301 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc 3361 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 3421 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa 3481 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct 3541 cgttgagctt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg 3601 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg 3661 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg 3721 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga 3781 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg 3841 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc 3901 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg 3961 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct 4021 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc 4081 gcacccgtgg ccaggaccca acgctgcccg agatctcgat cccgcgaaat //