LOCUS Exported 8262 bp ds-DNA circular SYN 17-JUN-2022 DEFINITION Gateway destination vector for bacterial expression; His tag. ACCESSION . VERSION . KEYWORDS pDest-527 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8262) TITLE Gateway Destination vectors for E. coli expression REFERENCE 2 (bases 1 to 8262) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 17, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8262 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(178..1038) /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 801..820 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1039..1131) /gene="bla" /label=AmpR promoter rep_origin 1212..1800 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 1986..2125 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2051..2070) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2211..2233 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2227..2418) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(2990..4072) /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 4034..4053 /label=LacI-R /note="LacI, reverse primer" promoter complement(4073..4150) /gene="lacI" /label=lacI promoter primer_bind 4356..4375 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4463..4482 /label=T7 /note="T7 promoter, forward primer" promoter 4463..4481 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4482..4506 /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 4521..4543 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4566..4583 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" protein_bind 4596..4720 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 4745..4775 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 4829..5488 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4895..4914) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" rep_origin 5541..6129 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS 6761..7066 /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 6945..6964 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(7107..7231) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 7291..7323 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 7330..7347 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 7513..7599 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 7691..7718 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" primer_bind complement(7772..7790) /label=T7 Term /note="T7 terminator, reverse primer" terminator 7786..7833 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 7870..63 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7957..7976) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8167..8188 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 61 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 121 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 181 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 241 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 301 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 361 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 421 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 481 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 541 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 601 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 661 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 721 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 781 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 841 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 901 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 961 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 1021 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt 1081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg 1141 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1201 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1261 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1321 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1381 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1441 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1501 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1561 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1621 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1681 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1741 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1801 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 1861 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 1921 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 1981 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt 2041 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc 2101 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg 2161 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 2221 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc 2281 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt 2341 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt 2401 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat 2461 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg 2521 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa 2581 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag 2641 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt 2701 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga 2761 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc 2821 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt 2881 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg 2941 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc 3001 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 3061 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca 3121 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt 3181 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt 3241 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg 3301 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa 3361 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc 3421 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca 3481 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac 3541 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac 3601 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag 3661 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc 3721 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca 3781 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt 3841 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc 3901 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca 3961 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct 4021 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca 4081 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt 4141 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc 4201 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg 4261 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc 4321 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca 4381 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag 4441 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac 4501 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atgagatctg 4561 gatcccacca tcaccatcac catagatctg atatcacaag tttgtacaaa aaagctgaac 4621 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga 4681 ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc 4741 aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc 4801 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt 4861 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg 4921 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa 4981 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc 5041 ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg 5101 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga 5161 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct 5221 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt 5281 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt 5341 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt 5401 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta 5461 ctgcgatgag tggcagggcg gggcgtaaac gcgtcgttcc actgagcgtc agaccccgta 5521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 5581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 5641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 5701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 5761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 5821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 5881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 5941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6181 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 6241 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 6301 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 6361 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgaac gcaattaata 6421 cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt 6481 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat 6541 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat 6601 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt 6661 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa 6721 atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac 6781 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac 6841 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc 6901 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga 6961 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga 7021 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct 7081 tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt 7141 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg 7201 tttctcgttc agctttcttg tacaaagtgg tgatatcgct agctagatag ctagaagctt 7261 gcggccgcac agctgtatac acgtgcaagc cagccagaac tcgctcctga agacccagag 7321 gatctcgagc accaccacca ccaccactaa tgttaattaa gttgggcgtt cctaggctga 7381 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact 7441 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga 7501 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc 7561 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac 7621 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat 7681 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg 7741 tttatttttc taaatacatt caaatatgta tccgctgagc aataactagc ataacccctt 7801 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg 7861 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 7921 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 7981 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 8041 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 8101 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 8161 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 8221 ttgatttata agggattttg ccgatttcgg cctattggtt aa //