LOCUS Exported 5807 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses mKelly1 tagged N-terminally with an MHHHHHHGSG tag. ACCESSION . VERSION . KEYWORDS pET-mKelly1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5807) AUTHORS Wannier TM, Gillespie SK, Hutchins N, McIsaac RS, Wu SY, Shen Y, Campbell RE, Brown KS, Mayo SL TITLE Monomerization of far-red fluorescent proteins. JOURNAL Proc Natl Acad Sci U S A. 2018 Nov 27;115(48):E11294-E11301. doi: 10.1073/pnas.1807449115. Epub 2018 Nov 13. PUBMED 30425172 REFERENCE 2 (bases 1 to 5807) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5807 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 550..569 /label=LacI-R /note="LacI, reverse primer" promoter complement(589..666) /gene="lacI" /label=lacI promoter primer_bind 872..891 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 979..998 /label=T7 /note="T7 promoter, forward primer" promoter 979..997 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 998..1022 /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 1037..1059 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1069..1086 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1783..1806 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" primer_bind complement(1833..1851) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1847..1894 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1931..2386 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2018..2037) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2228..2249 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2501..3361) /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 3124..3143 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3362..3454) /gene="bla" /label=AmpR promoter rep_origin 3535..4123 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4024..4043 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4277..4294 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4309..4448 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4374..4393) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4534..4556 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4550..4741) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5313..588) /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 MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEYSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa 61 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt 121 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc 181 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg 241 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga 301 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga 361 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc 421 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt 481 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata 541 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct 601 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat 661 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 721 gtaggttgag gccgttgagc accgccaccg caaggaatgg tgcatgcaag gagatggcgc 781 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga 841 gcccgaagtg gcgagcccga tcttccccat aggtgatgtc ggcgatatag gcgccagcaa 901 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg 961 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt 1021 cccctctaga aataattttg tttaacttta agaaggagat ataccatgca ccatcatcac 1081 caccacggtt ctggtgaact gattaaagaa aacatgcgta tgaaactgta catggaaggt 1141 accgttggca accatcactt caaatgcacc gcggaaggtg agggtaaacc gtacgaaggc 1201 acccagaccc agcgtattaa agttgttgaa ggtggtccgc tgccgttcgc gttcgacatc 1261 ctggcgacct gcttcatgta tggttctaaa acttttatca accaccctca gggtatcccg 1321 gacttcttca aacagtcttt cccagaaggt ttcacctggg aacgtgttac cacctacgaa 1381 gacggcggtg ttctgaccgt tacccaggac acctctctgc aggacggtta cctgatctac 1441 aacgttaaac tgcgtggtgt taacttcccg tctaacggcc ctgttatgca gaaaaagact 1501 ctgggttggg aaccgaccac cgagacactg gtcccggttg acggtggcct ggtgggtaga 1561 tgtgatatgg cgctgcgtct ggttggtggc ggtcacctga agtgcaatct gaaaaccacg 1621 tatcgttcca aaaaaccagc gaaaaacctg aagatgccgg gtaagtacca ggttgaccgt 1681 aagctggagc gtatcaaaga ggcggacaac gaaacctacg ttgaacagca cgaagttgcg 1741 gttgcgagat aatgagcttt cttgtacaaa gtggtttccg cttggagcca cccgcagttc 1801 gaaaagtaag tgattaacct aggctgctgc caccgctgag caataactag cataacccct 1861 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt 1921 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 1981 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 2041 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 2101 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 2161 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 2221 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 2281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 2341 caaaaattta acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg 2401 gcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 2461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 2521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 2581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 2641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 2701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 2761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 2821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 2881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 2941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 3001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 3061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 3121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 3181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 3241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 3301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 3361 tactcttcct ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga 3421 tacatatttg aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg 3481 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 3541 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 3601 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 3661 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 3721 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 3781 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 3841 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 3901 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 3961 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 4021 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 4081 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 4141 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 4201 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 4261 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta 4321 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc 4381 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca 4441 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 4501 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 4561 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa 4621 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg 4681 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca 4741 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag 4801 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg 4861 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat 4921 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga 4981 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa 5041 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt 5101 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc 5161 agcctagccg ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc 5221 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat 5281 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 5341 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 5401 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc 5461 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa 5521 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtattcggt atcgtcgtat 5581 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg 5641 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc 5701 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc 5761 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacg //