LOCUS Exported 6496 bp ds-DNA circular SYN 03-SEP-2019 DEFINITION Vector for in vivo biotinylation of a fusion protein in bacteria.. ACCESSION . VERSION . KEYWORDS MAK463 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6496) AUTHORS Tonia Rex TITLE Tonia Rex lab plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 6496) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Feb 27, 2020 from SnapGene 5.0.7 https://www.snapgene.com FEATURES Location/Qualifiers source 1..6496 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 85..107 /label=pGEX 3' /note="pGEX vectors, reverse primer" /note="color: ?; sequence: CCGGGAGCTGCATGTGTCAGAGG" CDS complement(101..292) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /note="color: #993366" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" promoter 788..865 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." /note="color: #ffffff; direction: RIGHT" CDS 866..1948 /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)." /note="color: #993366" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(885..904) /label=LacI-R /note="LacI, reverse primer" /note="color: ?; sequence: GGCATACTCTGCGACATCGT" promoter 2191..2219 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" /note="This forward directional feature has 3 segments: 1: 2191 .. 2196 / #ffffff / -35 2: 2197 .. 2212 / #ffffff 3: 2213 .. 2219 / #ffffff / -10" protein_bind 2227..2243 /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)." /note="color: #31849b" primer_bind 2232..2254 /label=M13/pUC Reverse /note="In lacZ gene" /note="color: ?; sequence: AGCGGATAACAATTTCACACAGG" primer_bind 2241..2264 /label=pMALseqF1 /note="color: ?; sequence: caatttcacacaggaaacagccag; added: 2019-04-18" primer_bind 2277..2305 /label=MAK418F /note="color: ?; sequence: ttttcacgagcaattgaccaacaaggacc; added: 2019-03-08" CDS 2313..2396 /codon_start=1 /gene="malE" /product="maltose binding protein from E. coli" /label=MBP /note="This gene encodes a signal sequence that can direct MBP to the bacterial periplasm." /note="This feature has 2 segments: 1: 2313 .. 2390 / #cc99b2 / signal sequence 2: 2391 .. 2396 / #cc99b2 Cleavage site after base 2390" /translation="MKIKTGARILALSALTTMMFSASALAKI" primer_bind complement(2377..2409) /label=MAK436R /note="color: ?; sequence: gcacctgggatccgattttggcgagagccgagg; added: 2019-04-18" primer_bind 2387..2420 /label=MAK442F /note="color: ?; sequence: cgccaaaatcggatcccaggtgcaattggtagag; added: 2019-05-08" CDS 2403..2786 /codon_start=1 /label=TP1170@MsIgK /note="color: #993366" /translation="QVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGR EWVAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCAKTYSGNY YSNYTVANYGTTGRGTLVTVSSGG" misc_feature 2478..2507 /label=CDR1 /note="color: #993366; direction: RIGHT" misc_feature 2550..2579 /label=CDR2 /note="color: #993366; direction: RIGHT" misc_feature 2697..2747 /label=CDR3 /note="color: #993366; direction: RIGHT" primer_bind complement(2765..2804) /label=MAK441R /note="color: ?; sequence: gtcgttcagtccgatatctccaccggaggagactgtgacc; added: 2019-05-09" primer_bind 2774..2793 /label=MAK461F /note="color: ?; sequence: ctcctccggtggagatatcg; added: 2019-06-18" CDS 2793..2834 /codon_start=1 /label=AviTAG /note="color: #993366" /translation="GLNDIFEAQKIEWH" primer_bind complement(2811..2877) /label=MAK461R /note="color: ?; sequence: ccttgtaatcgaattcttaGTGGTGATGATGGTGATGGGAGCCgtgccattcgattttc tgagcttc; added: 2019-06-18" CDS 2841..2858 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /note="color: #cc99b2" /translation="HHHHHH" CDS 2868..2891 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /note="color: #cc99b2" /translation="DYKDDDDK" terminator 2903..2989 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" /note="color: #ffffff" primer_bind complement(2965..2985) /label=pMALseqR /note="color: ?; sequence: gtcctactcaggagagcgttc" terminator 3081..3108 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" /note="color: #ffffff" promoter 3136..3226 /gene="bla" /label=AmpR promoter /note="color: #ffffff; direction: RIGHT" CDS 3227..4087 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: 1: 3227 .. 3295 / #ccffcc / signal sequence 2: 3296 .. 4087 / #ccffcc Cleavage site after base 3295" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3445..3464) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" /note="color: ?; sequence: ATAATACCGCGCCACATAGC" rep_origin 4175..4763 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" primer_bind 4664..4683 /label=pBR322ori-F /note="pBR322 origin, forward primer" /note="color: ?; sequence: GGGAAACGCCTGGTATCTTT" primer_bind 4929..4948 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" /note="color: ?; sequence: GGTGATGTCGGCGATATAGG" protein_bind 5051..5075 /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)." /note="color: #31849b" RBS 5090..5112 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" /note="color: #a6acb3" CDS 5119..6081 /codon_start=1 /gene="birA" /product="E. coli biotin protein ligase" /label=BirA /note="color: #993366" /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" CDS 6120..6137 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /note="color: #cc99b2" /translation="HHHHHH" primer_bind complement(6190..6208) /label=T7 Term /note="T7 terminator, reverse primer" /note="color: ?; sequence: GCTAGTTATTGCTCAGCGG" terminator 6204..6251 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" /note="color: #ffffff" primer_bind 6381..6398 /label=L4440 /note="L4440 vector, forward primer" /note="color: ?; sequence: AGCGAGTCAGTGAGCGAG" ORIGIN 1 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 61 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 121 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac 181 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct 241 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct 301 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc 361 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac 421 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct 481 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc 541 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac 601 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg 661 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc 721 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg 781 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga 841 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 901 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 961 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 1021 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 1081 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 1141 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 1201 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 1261 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 1321 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 1381 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 1441 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 1501 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 1561 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 1621 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatattt cggtagtggg 1681 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 1741 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc 1801 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc 1861 caatacacaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 1921 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc 1981 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc 2041 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata 2101 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa 2161 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt 2221 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt 2281 cacgagcaat tgaccaacaa ggaccataga ttatgaaaat aaaaacaggt gcacgcatcc 2341 tcgcattatc cgcattaacg acgatgatgt tttccgcctc ggctctcgcc aaaatcggat 2401 cccaggtgca attggtggag tctgggggag gctgggtgca gcctgggggg tctctgagac 2461 tctcctgtgc agcctctgga ttcaccttca gtgacactgc catgatgtgg gtccgccagg 2521 ctccagggaa ggggcgagag tgggtcgcag ctattgatac tggtggtgga tacacatact 2581 atgcagactc cgtgaagggc cgattcacca tctccagaga caacgccaag aacacgctgt 2641 atctgcaaat gaacagcctg aaaccggagg acacggcccg atactactgt gcgaagacct 2701 actccggtaa ttactatagc aactacacgg ttgccaacta tggcaccacg ggccggggga 2761 ccctggtcac agtctcctcc ggtggagata tcggactgaa cgacatcttc gaagctcaga 2821 aaatcgaatg gcacggctcc catcaccatc atcaccacta agaattcgat tacaaggatg 2881 acgacgataa gtaaataagc ttcaaataaa acgaaaggct cagtcgaaag actgggcctt 2941 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc 3001 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 3061 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 3121 aactctttcg gtccgttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 3181 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 3241 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 3301 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 3361 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc 3421 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 3481 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 3541 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 3601 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 3661 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 3721 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 3781 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 3841 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 3901 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 3961 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 4021 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 4081 ttggtaactg tcagaccaag tttactcata tatactttag attgatttcc ttaggactga 4141 gcgtcaaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4201 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4261 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4321 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4381 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4441 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4501 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 4561 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 4621 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 4681 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 4741 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 4801 tttgctggcc ttttgctcac atgttccggt accgcgccca acagtccccc ggccacgggg 4861 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 4921 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 4981 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac 5041 tcactagagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact 5101 ttaagaagga gatataccat gaaggataac accgtgccac tgaaattgat tgccctgtta 5161 gcgaacggtg aatttcactc tggcgagcag ttgggtgaaa cgctgggaat gagccgggcg 5221 gctattaata aacacattca gacactgcgt gactggggcg ttgatgtctt taccgttccg 5281 ggtaaaggat acagcctgcc tgagcctatc cagttactta atgctaaaca gatattgggt 5341 cagctggatg gcggtagtgt agccgtgctg ccagtgattg actccacgaa tcagtacctt 5401 cttgatcgta tcggagagct taaatcgggc gatgcttgca ttgcagaata ccagcaggct 5461 ggccgtggtc gccggggtcg gaaatggttt tcgccttttg gcgcaaactt atatttgtcg 5521 atgttctggc gtctggaaca aggcccggcg gcggcgattg gtttaagtct ggttatcggt 5581 atcgtgatgg cggaagtatt acgcaagctg ggtgcagata aagttcgtgt taaatggcct 5641 aatgacctct atctgcagga tcgcaagctg gcaggcattc tggtggagct gactggcaaa 5701 actggcgatg cggcgcaaat agtcattgga gccgggatca acatggcaat gcgccgtgtt 5761 gaagagagtg tcgttaatca ggggtggatc acgctgcagg aagcggggat caatctcgat 5821 cgtaatacgt tggcggccat gctaatacgt gaattacgtg ctgcgttgga actcttcgaa 5881 caagaaggat tggcacctta tctgtcgcgc tgggaaaagc tggataattt tattaatcgc 5941 ccagtgaaac ttatcattgg tgataaagaa atatttggca tttcacgcgg aatagacaaa 6001 cagggggctt tattacttga gcaggatgga ataataaaac cctggatggg cggtgaaata 6061 tccctgcgta gtgcagaaaa aggtaagctt tgatgaggac tagtgcggcc gcactcgagc 6121 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg 6181 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga 6241 ggggtttttt gctgaaagga ggaactatat ccggatgcta gcacatgttc tttcctgcgt 6301 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6361 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc 6421 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catataaggt gcactgtgac 6481 tgggtcatgg ctgcgc //