LOCUS Exported 5810 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 5810) 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 5810) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5810 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(582..1442) /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 1205..1224 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1443..1535) /gene="bla" /label=AmpR promoter rep_origin 1616..2204 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2105..2124 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2358..2375 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2390..2532 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2458..2477) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2618..2640 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2634..2825) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3397..4479) /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 4441..4460 /label=LacI-R /note="LacI, reverse primer" promoter complement(4480..4557) /gene="lacI" /label=lacI promoter primer_bind 4763..4782 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4870..4889 /label=T7 /note="T7 promoter, forward primer" promoter 4870..4888 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4889..4913 /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 4928..4950 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4960..4977 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5674..5697 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" primer_bind complement(5724..5742) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5738..5785 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat 481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg 1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac 1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac 2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg 2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg 2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga 2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa 2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt 2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt 3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct 3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac 3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag 3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc 3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc 3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc 3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt 3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg 3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc 3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat 3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt 3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc 3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc 3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag 3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt 4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat 4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac 4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg 4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg 4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt 4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc 4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact 4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg 4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga 4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc 4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt 4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg 4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc 4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 4921 aaataatttt gtttaacttt aagaaggaga tataccatgc accatcatca ccaccacggt 4981 tctggtgaac tgattaaaga aaacatgcgt atgaaactgt acatggaagg taccgttggc 5041 aaccatcact tcaaatgcac cgcggaaggt gagggtaaac cgtacgaagg cacccagacc 5101 cagcgtatta aagttgttga aggtggtccg ctgccgttcg cgttcgacat cctggcgacc 5161 tgcttcatgt atggttctaa aacttttatc aaccaccctc agggtatccc ggacttcttc 5221 aaacagtctt tcccagaagg tttcacctgg gaacgtgtta ccacctacga agacggcggt 5281 gttctgaccg ttacccagga cacctctctg caggacggtt acctgatcta caacgttaaa 5341 ctgcgtggtg ttaacttccc gtctaacggc cctgttatgc agaaaaagac tctgggttgg 5401 gaaccgacca ccgagacact ggtcccggtt gacggtggcc tggtgggtag atgtgatatg 5461 gcgctgcgtc tggttggtgg cggtcacctg aagtgcaatc tgaaaaccac gtatcgttcc 5521 aaaaaaccag cgaaaaacct gaagatgccg ggtaagtacc aggttgaccg taagctggag 5581 cgtatcaaag aggcggacaa cgaaacctac gttgaacagc acgaagttgc ggttgcgaga 5641 taatgagctt tcttgtacaa agtggtttcc gcttggagcc acccgcagtt cgaaaagtaa 5701 gtgattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc 5761 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat //