LOCUS Exported 9122 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Analogous to pMINTNH3 but with a C-terminal 3C cleavage site and a GFP-His10 tag fusion.. ACCESSION . VERSION . KEYWORDS pMINTC3GH (Hyg) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9122) TITLE A uniform cloning platform for mycobacterial genetics and protein production REFERENCE 2 (bases 1 to 9122) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9122 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 162..179 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind complement(298..322) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS complement(401..706) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(503..522) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1048..1707) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 1622..1641 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(1761..1791) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(1816..1940) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(1959..1979) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1983..2003) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2030..2053 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 2060..2770 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" primer_bind complement(2087..2115) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 2710..2730 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" CDS 2777..2806 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" rep_origin complement(3076..3664) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3156..3175) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5668..5687 /label=pENTR-R /note="pENTR vectors, reverse primer" terminator 7623..7669 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 7925..7947 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7944..7960 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7944..7960 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 7980..8000 /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" primer_bind 8053..8069 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 8054..8070 /label=KS primer /note="common sequencing primer, one of multiple similar variants" protein_bind 9007..9031 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" ORIGIN 1 gtcggcaccg tcacggccgt gggaggcggc acgatccgcg acgtgatgat cggccgcatc 61 cccacggtgc tgcgcagtga gctctacgcc atcccggcgt tgatctgtgc gttcgcacgc 121 acaggcccgg tgtgagaagg gtctctgacg agcgggagaa ctccctatca gtgatagagt 181 ttgtcctccc tatcagtgat agataggctc tgggagtacc cgtgtgtacg accagcacgg 241 catacatcat ttcgacgccg agagattcgc cgcccgaaat gagcacgatc cgcatgcacc 301 cagctttctt gtacaaagtg ggctcgaggc atgcgaagga gatatacata tgagttgaag 361 agcgacctgc agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca 421 ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga 481 taacggagac cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga 541 tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca 601 gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca 661 gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagcccctg 721 ttctcgtcag caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc 781 tgattttccg ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg 841 cttaccagac cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt 901 caactgtcac tgtaatacgc tgcttcatag catacctctt tttgacatac ttcgggtata 961 catatcagta tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct 1021 ggcttttagt aagccggatc cacgcgttta cgccccgccc tgccactcat cgcagtactg 1081 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg 1141 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac 1201 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca 1261 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt 1321 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg 1381 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg 1441 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg 1501 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt 1561 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg 1621 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt 1681 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgacgg 1741 atcctaactc aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt 1801 gcctaatgcg gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt 1861 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca 1921 gcttttttgt acaaacttgt gatatcgaat tcgctagcta cctttctctt cttttttgga 1981 tctacctttc tcttcttttt tggggatcca ctagttcagc tcttctgcat tagaagtttt 2041 gtttcaaggt ccacaattca gcaaaggaga agaacttttc actggagttg tcccaattct 2101 tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg 2161 tgatgctaca tacggaaaac tcacccttaa atttatttgc actactggaa aactacctgt 2221 tccttggcca acacttgtca ctactctgac ctatggtgtt caatgctttt cccgttatcc 2281 ggatcacatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga 2341 acgcactata tctttcaaag atgacgggaa ctacaagacg cgtgctgaag tcaagtttga 2401 aggtgatacc cttgttaatc gtatcgagtt aaagggtatt gattttaaag aagatggaaa 2461 cattctcgga cacaaactag agtacaacta taactcacac aatgtataca tcacggcaga 2521 caaacaaaag aatggaatca aagctaactt caaaattcgc cacaacattg aagatggttc 2581 cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt 2641 accagacaac cattacctgt cgacacaatc tgccctttcg aaagatccca acgaaaagcg 2701 tgaccacatg gtccttcttg agtttgtaac tgctgctggg attacacatg gcatggatga 2761 gctctacaaa actagtcatc atcatcacca tcatcatcac catcattaat tatacatagt 2821 tgcttaagct gcaggggacc ctagaggtcc ccttttttat tttaaaaatt ttttataacc 2881 ggtaagaagt ccaacacgcc gctcctcccc gatgttgcgt agccgcttca agcttaagca 2941 acggccccag tgagtagcga ctttggatcc ggtacccgca acggtcaacc agcgtctacg 3001 cgtttcactg gccgacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3061 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3121 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3181 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3241 tctcccctcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 3301 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3361 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 3421 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 3481 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 3541 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 3601 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 3661 tcaagaagat cctttgatct tttctacggg gtctgacgct catggaacta gtgaggtctg 3721 cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag 3781 aaagtgaggg agccacggtt ggtgagagct ttgttgtagg tggaccagtt ggtgattttg 3841 aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc 3901 aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc 3961 tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat 4021 gaaactgcaa gtattctaga ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4081 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 4141 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 4201 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 4261 cgacgtggcc gaccagcccg tcatcgtcaa cgcctgatcc gcggtgcgga caggccgtgt 4321 cgcgaccggc cgtgcggaat taagccggcc cgtaccctgt gaatagaggt ccgctgtgac 4381 acaagaatct ctgttacttc tcgaccgtat tgattcggat gattcctacg cgagcctgcg 4441 gaacgaccag gaattctggg agccgctggc ccgccgagcc ctggaggagc tcgggctgcc 4501 ggtgccgccg gtgctgcggg tgcccggcga gagcaccaac cccgtactgg tcggcgagcc 4561 cggcccggtg atcaagctgt tcggcgagca ctggtgcggt ccggagagcc tcgcgtcgga 4621 gtcggaggcg tacgcggtcc tggcggacgc cccggtgccg gtgccccgcc tcctcggccg 4681 cggcgagctg cggcccggca ccggagcctg gccgtggccc tacctggtga tgagccggat 4741 gaccggcacc acctggcggt ccgcgatgga cggcacgacc gaccggaacg cgctgctcgc 4801 cctggcccgc gaactcggcc gggtgctcgg ccggctgcac agggtgccgc tgaccgggaa 4861 caccgtgctc accccccatt ccgaggtctt cccggaactg ctgcgggaac gccgcgcggc 4921 gaccgtcgag gaccaccgcg ggtggggcta cctctcgccc cggctgctgg accgcctgga 4981 ggactggctg ccggacgtgg acacgctgct ggccggccgc gaaccccggt tcgtccacgg 5041 cgacctgcac gggaccaaca tcttcgtgga cctggccgcg accgaggtca ccgggatcgt 5101 cgacttcacc gacgtctatg cgggagactc ccgctacagc ctggtgcaac tgcatctcaa 5161 cgccttccgg ggcgaccgcg agatcctggc cgcgctgctc gacggggcgc agtggaagcg 5221 gaccgaggac ttcgcccgcg aactgctcgc cttcaccttc ctgcacgact tcgaggtgtt 5281 cgaggagacc ccgctggatc tctccggctt caccgatccg gaggaactgg cgcagttcct 5341 ctgggggccg ccggacaccg cccccggcgc ctgacgcccc gggcccgcgc cgaagcccgc 5401 tgctgcgagc ccggagcggg ccggccgacg gcggtgcggg cccggcggcg gacgctcagc 5461 agcggcgggc gtgaaaggcc ctggcatcct cgatcatctc ctccagggtg ggtcggccgg 5521 gcttccatcc cagctcggca aggatcaacg aggacagtcg cacgacgaag ttcttctgga 5581 tcgcgcccgt gctggaagca ctcaacctcg aagcgtgtgg ttgcggagcc atctagcaac 5641 cacacgacca tggtactccc gttgaatatg gctcataaca ccccttgtat tactgtttat 5701 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca 5761 gagattttga gacacaacgt ggctttcccc ccccccgacg tcaggtggct agctggcggg 5821 aacggcaatg ctcctggcgg cggcggggct ggtgcccagg tctccgcaca gagcggcggt 5881 gctggcgctc gcggccaggc gtggttcttc gcgtactgac aagaaacccc cctctttagg 5941 actcagtgtc cttgggaggg gggctttttg cgtttcagga ggtcttggcc agcttggaca 6001 tcgcctcagc gatagcctcg tcgcgggcct cagacgccat ctggtacttc atcgccatcc 6061 taggagtcgt gtgaccgaga cgggccatca gctccttggt cgtcgcacct gcctgagcgg 6121 cgaacgtagc gccgacagcg cggaggtcgt ggatgcggag ttccggccga ccgatcttgg 6181 cgtagccacg cttcagcgac ttggtgaacg cggacttcga cagccggttg ccctgcgtcg 6241 tggtcaccag gaatgcctcg gggcccttgt tcatcttcgt acggtccttc atgtgcgctc 6301 ggatcatctc cgcgacgtga ggcggaaccg tcacaggacg cttcgaccgg acggtcttgg 6361 cgttgccaac gacgatcttg ttccccacgc gggaagcgcc acggcgcacc cggagcttca 6421 tcgtcatgcc gtcgtccacg atgtccttgc ggcgaagctc gatcagctct ccgaaccgga 6481 ggctcgtcca cgccaggatg tatgccgcga tccggtagtg cacgaagatc tcagcggcga 6541 cgatgtccag ctcctcaggc gtcagcgcct ctacgtcgcg ctcatcggct gccttctgct 6601 cgatccggca cgggttctct gcgatcagct tgtcctcgac cgctgtgttc atcaccgccc 6661 ggaggacgtt gtaggcatgc cggcgggcag tcgggtgctt cctacccatc ccggcccacc 6721 acgcacgcac cagagctggc gtcatctctg tgaccgccac ttcacctagc accgggtaga 6781 tgcggcgctc cgcgtgcccg ctgtacagat ccctggtgcc gtctgcgagg tcgcgctcca 6841 cgagccactt ccgggtgtac tcctccagcg tgatggcgct ggcggctgcc ttcttcgccc 6901 ggtcctgtgg aggggtccag gtctccatct cgatgagccg cttctcgccc gcgagccagg 6961 cttcggcgtc catcttgttg tcgtaggtct gcagcgcgta gtacctcaca ccgtcctgcg 7021 ggttgacgta tgaggcttgg atcctcccgc tgcgctgagt cttcagcgat ccccatccgc 7081 gacgtgccaa ctaggtctcc tctcgtcgtg aacaaggcta ccgggttgca actcctgtgc 7141 aactctcagg cttcaacgcg cttctacgac ctgcaatttc tttccactta gaggatgcag 7201 ccgagagggg gtaaaaacct atcttgaccg gcccatatat gtggtcggca gacacccatt 7261 cttccaaact agctacgcgg gttcgattcc cgtcgcccgc tccgctggtc agagggtgtt 7321 ttcgccctct ggccattttt ctttccaggg gtctgcaact cttgtgcgac tcttctgacc 7381 tgggcatacg cggttgcaac gcatccctga tctggctact ttcgatgctg acaaacgaat 7441 agagcccccc gcctgcgcga acagacgagg ggcattcaca ccagattgga gctggtgcag 7501 tgaagagaat agaccgggac aaggttgcac cgggagttgc agcggtcgga accctcgccg 7561 tcggcgggct ggcgttcgcc ctgtcgttca cggctctagc tgatcaccgc ggccatgatg 7621 gcataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag 7681 tcgatcgtac gctagttaac tacgtcgaca tcgatttaat taatatgcat caattgattt 7741 aaatactagt gttaactatt taattgggga ccctagaggt cccctttttt attttaaaaa 7801 ttttttcaag gggacctcta gggtccccag ctggctagcg agtcatgagg tcaccatgtt 7861 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 7921 taccagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctatc 7981 aactttgtat agaaaagttg catgaccaac ttcgataacg ttctcggctc gatgatcccc 8041 gggggccccc cctcgaggtc gacggtatcg atttaattaa tatgcatcaa ttgatttaaa 8101 tactagtgtt aactatttaa ttggggaccc tagaggtccc cttttttatt ttaaaaattt 8161 tttcaagggg acctctaggg tccccagctg gctagcgagt catgaggtca ccatggtcat 8221 taggagccgc tctcgcactt cagctgcttc tccaagccgc agatgatcag ctccaggccg 8281 aacaagaagg ccggctcggc gccctggtgg tcgaacagct cgatggcctg gcgcagcaac 8341 ggcggcatgc tgtcggtggt cggggtctcg cgctcctcct tggcgacctg gtgctcctgg 8401 tcctccaaca cgcagcccag ggtgaagtgg cccacggccg acagggcgta caacgcgttc 8461 tccagcgaga agccctgctg gcacaggaag gccaactggt tctccagggt ctcgtactgc 8521 ttctcggtcg gccgggtgcc caggtgcacc ttggcgccgt cgcggtggct cagcaaggcg 8581 cagcggaagc tcttggcgtt gttgcgcagg aagtcctgcc agctctcgcc ctccagcggg 8641 cagaagtggg tgtggtggcg gtccaacatc tcgatggcca gggcgtcgag cagggcccgc 8701 ttgttcttga cgtgccagta cagggtcggc tgctcgacgc ccagcttctg ggccagcttg 8761 cgggtggtca ggccctcgat gccgacctcg ttcagcagct ccagggcgga gttgatgacc 8821 ttggacttgt ccaggcggga cattgcgaag tgattcctcc tgatggtacc ggatccgtgg 8881 caggagggtc gcctgcgtcg gcaactcgcc caggctacac gagagccctc gactgcgaaa 8941 atgccgacca ggcaccaccg cactccaggc ttaacccggt gcgacgatcc aatattacga 9001 gatctgacaa gtttgtacaa aaaagcaggc taccaggcct agatctgggg accctagagg 9061 tccccttttt tattttaaaa attttttcac gtgccagccg attggactag aaatattgga 9121 tc //