LOCUS Exported 5975 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pET28a expressing codon optimized superfolder GFP with C-terminal His6 tag. ACCESSION . VERSION . KEYWORDS pET28a-sfGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5975) AUTHORS Peeler JC, Mehl RA TITLE Site-specific incorporation of unnatural amino acids as probes for protein conformational changes. JOURNAL Methods Mol Biol. 2012;794:125-34. doi: 10.1007/978-1-61779-331-8_8. PUBMED 21956560 REFERENCE 2 (bases 1 to 5975) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5975 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 26..73 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 69..87 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(140..157) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(169..186) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(193..909) /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" RBS 916..938 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 953..977 /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)." primer_bind complement(977..996) /label=T7 /note="T7 promoter, forward primer" promoter complement(978..996) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1080..1099) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 1305..1382 /gene="lacI" /label=lacI promoter CDS 1383..2465 /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 complement(1402..1421) /label=LacI-R /note="LacI, reverse primer" CDS 3274..3465 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(3459..3481) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 3567..3706 /label=bom /note="basis of mobility region from pBR322" primer_bind 3622..3641 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(3721..3738) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3892..4480) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3972..3991) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(4591..4610) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 4602..5417 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(4675..4694) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(5509..5964) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5646..5667) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5858..5877 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctttaat ggtgatgatg 181 atggtggctg cctttataca gttcatccat accgtgggta atgcccgcgg cggtaacaaa 241 ttccagcagc accatatgat cacgtttttc attcggatct ttgctcagaa cgctctgggt 301 gctcagataa tgattatccg gcagcagcac cgggccatca ccaatcgggg tattctgctg 361 ataatgatcc gccagctgca cgctaccatc ttccacgttg tgacggattt taaagttcgc 421 tttgatgcca tttttctgtt tatcggcggt aatatacaca ttatggctgt tgaaattata 481 ttccagttta tgacccagaa tgttgccatc ttctttaaaa tcaatacctt tcagttcaat 541 gcggttcacc agggtatcgc cttcaaattt aacttccgca cgggttttat aggtgccatc 601 atctttgaag ctaatggtac gttcctgcac atagccttcc ggcatcgcgc ttttaaagaa 661 atcatggcgt ttcatatgat ccggatagcg gctaaagcac tgaacgccat aggtcagggt 721 ggtcaccagg gtcggccacg gaaccggcag tttaccggtg gtgcaaataa atttcagggt 781 cagtttaccg ttggtcgcat caccttcgcc ttcgccacga acgctaaatt tatggccatt 841 cacatcacca tccagttcca ccagaatcgg cacaacgccg gtaaacagtt cttcaccttt 901 gctaaccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt 961 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat 1021 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc 1081 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc 1141 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc 1201 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg 1261 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc 1321 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga 1381 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg 1441 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag 1501 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac 1561 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg 1621 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag 1681 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca 1741 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct 1801 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta 1861 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc 1921 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg 1981 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact 2041 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca 2101 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt 2161 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct 2221 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca 2281 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc 2341 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc 2401 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 2461 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg 2521 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 2581 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 2641 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg 2701 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc 2761 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg 2821 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag 2881 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc 2941 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag 3001 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt 3061 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg 3121 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt 3181 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc 3241 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt 3301 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg 3361 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac 3421 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 3481 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 3541 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga 3601 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg 3661 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 3721 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 3781 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 3841 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 3901 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 3961 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4021 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4081 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4141 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4201 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4261 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4321 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4381 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4441 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4501 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa 4561 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg 4621 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata 4681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc 4741 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag 4801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt 4861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat 4921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt 4981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat 5041 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg 5101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc 5161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg 5221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc 5281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc 5341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc 5401 tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa 5461 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg 5521 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 5581 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 5641 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 5701 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 5761 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag 5821 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 5881 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 5941 ttaatgcgcc gctacagggc gcgtcccatt cgcca //