LOCUS Exported 5807 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses mKelly2 tagged N-terminally with an MHHHHHHGSG tag. ACCESSION . VERSION . KEYWORDS pET-mKelly2 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 921..940 /label=LacI-R /note="LacI, reverse primer" promoter complement(960..1037) /gene="lacI" /label=lacI promoter primer_bind 1243..1262 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1350..1369 /label=T7 /note="T7 promoter, forward primer" promoter 1350..1368 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1369..1393 /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 1408..1430 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1440..1457 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2154..2177 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" primer_bind complement(2204..2222) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2218..2265 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2302..2757 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2389..2408) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2599..2620 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2872..3732) /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 3495..3514 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3733..3825) /gene="bla" /label=AmpR promoter rep_origin 3906..4494 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4395..4414 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4648..4665 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4680..4819 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4745..4764) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4905..4927 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4921..5112) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5684..959) /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" ORIGIN 1 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 121 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg 181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca 1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata 1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag 1321 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgc 1441 accatcatca ccaccacggt tctggtgaac tgattaaaga aaacatgcgt atgaaactgt 1501 acatggaagg taccgttggc aaccatcact tcaaatgcac cgcggaaggt gagggtaaac 1561 cgtacgaagg cacccagacc cagcgtatta aagttgttga aggtggtccg ctgccgttcg 1621 cgttcgacat cctggcgacc tgcttcatgt atggttctaa aacttttatc aactaccctc 1681 agggtatccc ggacttcttc aaacagtctt tcccagaagg tttcacctgg gaacgtgtta 1741 ccacctacga agacggcggt gttctgaccg ttacccagga cacctctctg caggacggtt 1801 acctgatcta caacgttaaa ctgcgtggtg ttaacttccc gtctaacggc cctgttatgc 1861 agaaaaagac tctgggttgg gaaccgacca ccgagtacct ggtcccggtt gacggtggcc 1921 tggagggtac atgtgatatg gcgctgcgtc tggttggtgg cggtcacctg aagtgcaatc 1981 tgaaaaccac gtatcgttcc aaaaaaccag cgaaaaacct gaagatgccg ggtgagcacc 2041 aggttgaccg taagctggag cgtatcaaag aggcggacaa cgaaacctac gttgaacagc 2101 acgaagttgc ggttgcgaga taatgagctt tcttgtacaa agtggtttcc gcttggagcc 2161 acccgcagtt cgaaaagtaa gtgattaacc taggctgctg ccaccgctga gcaataacta 2221 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 2281 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 2341 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 2401 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 2461 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 2521 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 2581 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 2641 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 2701 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg 2761 gcggcacgat ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 2821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 2881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 2941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3721 ttgaatactc atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc 3781 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa 3841 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 3901 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 3961 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 4021 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 4081 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 4141 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 4201 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 4261 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 4321 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 4381 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 4441 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 4501 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 4561 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 4621 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 4681 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc 4741 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg 4801 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 4861 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 4921 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 4981 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 5041 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 5101 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc 5161 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact 5221 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac 5281 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca 5341 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag 5401 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 5461 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 5521 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc 5581 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc 5641 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca 5701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 5761 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacg //