LOCUS Exported 6559 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for recombinant bacterial expression of SnoopLigase, a protein that promotes the formation of an isopeptide bond between 2 peptide tags (SnoopTagJr and DogTag), with N-terminal HaloTag7. ACCESSION . VERSION . KEYWORDS pET28a-HaloTag7-SnoopLigase SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6559) AUTHORS Buldun CM, Jean JX, Bedford MR, Howarth M TITLE SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. JOURNAL J Am Chem Soc. 2018 Feb 28;140(8):3008-3018. doi: 10.1021/jacs.7b13237. Epub 2018 Feb 14. PUBMED 29402082 REFERENCE 2 (bases 1 to 6559) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6559 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 126..145 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 229..248 /label=T7 /note="T7 promoter, forward primer" promoter 229..247 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 248..272 /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)." RBS 287..309 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 328..345 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 355..1239 /codon_start=1 /product="modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives" /label=HaloTag(R) /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG SEIARWLSTLEISG" primer_bind complement(1721..1739) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1735..1782 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1819..2274 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1906..1925) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2116..2137 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2366..3181) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 3089..3108 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3173..3192 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3303..3891 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3792..3811 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4045..4062 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4077..4216 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4142..4161) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4302..4324 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4318..4509) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5318..6400) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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" primer_bind 6362..6381 /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 61 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc 121 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg 181 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca 241 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta 301 agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag cggcgaaatc 361 ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac 421 gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc 481 tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca 541 gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac 601 gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt 661 cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa 721 ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt 781 gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat 841 cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc 901 gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc 961 ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa 1021 tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc 1081 gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct 1141 gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc 1201 gagatcgcgc gctggctgtc tactctggag atttccggtg gtagcggtgg gtctggtagt 1261 ggtgtgaata agaacgataa aaagccgctg cgtggtgccg tgtttagcct gcagaaacag 1321 catcccgact atcccgatat ctatggcgcg attgatcaga atgggaccta tcaaaatgtg 1381 cgtaccggcg aagatggtaa actgaccttt aagaatctga gcgatggcaa atatcgcctg 1441 tttgaaaata gcgaaccccc gggctataaa ccggtgcaga ataagccgat tgtggcgttt 1501 cagattgtga atggcgaagt gcgtgatgtg accagcattg tgccgccggg tgtgccggct 1561 acatatgaat ttacctaata aaacggtaaa cattatatca ccaatgaacc gataccgccg 1621 aaataataaa agcttgcggc cgcactcgag cactaccacc accaccactg agatccggct 1681 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 1741 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 1801 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 1861 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 1921 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 1981 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2041 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2101 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2161 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2221 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg 2281 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 2341 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca 2401 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 2461 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 2521 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 2581 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 2641 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 2701 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 2761 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 2821 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 2881 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 2941 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 3001 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 3061 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 3121 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca 3181 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc 3241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 3301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 3361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 3421 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 3481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 3541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 3601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 3661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 3721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 3781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 3841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 3901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 3961 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 4021 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg 4081 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca 4141 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga 4201 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 4261 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 4321 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg 4381 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc 4441 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg 4501 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat 4561 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga 4621 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca 4681 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca 4741 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact 4801 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca 4861 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca 4921 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc 4981 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac 5041 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt 5101 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc 5161 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc 5221 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc 5281 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg 5341 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 5401 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 5461 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 5521 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 5581 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 5641 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 5701 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 5761 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 5821 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 5881 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 5941 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 6001 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 6061 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 6121 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 6181 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 6241 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 6301 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 6361 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcgccacc tctgacttga 6421 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc 6481 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 6541 gaggccgttg agcaccgcc //