LOCUS Exported 6256 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET11a-Z-NspGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6256) AUTHORS Blakeley BD, Chapman AM, McNaughton BR TITLE Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo. JOURNAL Mol Biosyst. 2012 Aug;8(8):2036-40. Epub 2012 Jun 12. PUBMED 22692102 REFERENCE 2 (bases 1 to 6256) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6256 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 9..26 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 579..590 /codon_start=1 /product="WELQut protease recognition and cleavage site" /label=WELQut site /translation="WELQ" primer_bind complement(668..686) /label=T7 Term /note="T7 terminator, reverse primer" terminator 682..729 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(904..932) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(959..977) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1045..1149 /gene="bla" /label=AmpR promoter CDS 1150..2010 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1368..1387) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2181..2769 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2670..2689 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2923..2940 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2955..3097 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3023..3042) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3183..3205 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3199..3390) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4702..5784) /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 5746..5765 /label=LacI-R /note="LacI, reverse primer" promoter complement(5785..5862) /gene="lacI" /label=lacI promoter primer_bind 6068..6087 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6171..6190 /label=T7 /note="T7 promoter, forward primer" promoter 6171..6189 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6190..6214 /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 6229..6251 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" ORIGIN 1 atatgggtca tcaccaccac catcacggtg gcgctagcaa aggtgaacgt ctgtttcgtg 61 gtaaagtacc gatcttagtg gaattaaagg gcgacgtgaa cggtcataaa tttagcgtgc 121 gcggcgaagg caaaggtgac gctacccgtg gtaaattgac cctgaagttt atttgcacaa 181 caggcaaatt acccgttccg tggcccacct tagtgaccac cctgacctat ggcgttcagt 241 gcttcagtcg ttaccctaaa catatgaaac gtcacgattt tttcaaatca gccatgccta 301 aaggatatgt tcaagagcgt acaatcagct tcaagaagga tggcaaatat aaaacgcgtg 361 cggaagtgaa atttgaaggc cgcacactag taaatcgtat caaactgaaa ggtcgtgact 421 tcaaagaaaa aggcaacatt ttaggccata aactgcgtta taactttaat tctcataagg 481 tgtatattac ggccgataaa cgcggtggct ctggctctgg ctcgagcgcc ctcaaaaaag 541 aattgcaggc aaacaaaaaa gaacttgcgc agctgaagtg ggagttacaa gctctgaaaa 601 aggaactggc gcagtaagga tccggctgct aacaaagccc gaaaggaagc tgagttggct 661 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg 721 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg 781 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg 841 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac 901 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc 961 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 1021 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 1081 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 1141 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 1201 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 1261 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 1321 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 1381 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 1441 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 1501 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 1561 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 1621 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 1681 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 1741 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 1801 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 1861 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 1921 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 1981 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 2041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 2101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 2761 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 2821 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 2881 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 2941 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 3001 cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 3061 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 3121 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 3181 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct 3241 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc 3301 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat 3361 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt 3421 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga 3481 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg 3541 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt 3601 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc 3661 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc 3721 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc 3781 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac 3841 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct 3901 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct 3961 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg 4021 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag 4081 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg 4141 gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag ttaggctggt aagagccgcg 4201 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc 4261 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag 4321 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata 4381 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg 4441 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag 4501 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata 4561 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg 4621 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa 4681 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 4741 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 4801 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 4861 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 4921 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 4981 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 5041 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 5101 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 5161 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 5221 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 5281 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 5341 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 5401 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 5461 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 5521 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 5581 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 5641 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 5701 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 5761 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta 5821 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc 5881 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg 5941 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc 6001 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt 6061 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc 6121 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact 6181 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt 6241 taagaaggag atatac //