LOCUS Exported 5881 bp ds-DNA circular SYN 14-FEB-2022 DEFINITION Bacterial expression of membrane scaffold protein (MSP1D1) for Nanodisc formation. ACCESSION . VERSION . KEYWORDS pMSP1D1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5881) AUTHORS Denisov IG, Grinkova YV, Lazarides AA, Sligar SG TITLE Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. JOURNAL J Am Chem Soc. 2004 Mar 24. 126(11):3477-87. PUBMED 15025475 REFERENCE 2 (bases 1 to 5881) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 14, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5881 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 13..152 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(78..97) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 238..260 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(254..445) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(1254..2336) /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 2298..2317 /label=LacI-R /note="LacI, reverse primer" promoter complement(2337..2414) /gene="lacI" /label=lacI promoter primer_bind 2620..2639 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2723..2742 /label=T7 /note="T7 promoter, forward primer" promoter 2723..2741 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2742..2766 /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 2781..2803 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2816..2836 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" CDS 2858..2878 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3468..3485 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3538..3556) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3552..3599 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3636..4091 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3723..3742) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3933..3954 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4183..4998) /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 4906..4925 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4990..5009 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 5120..5708 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5609..5628 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5862..5879 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 61 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 121 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 181 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 241 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt 301 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca 361 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa 421 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg 481 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac 541 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc 601 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac 661 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact 721 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg 781 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct 841 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca 901 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg 961 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg 1021 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg 1081 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga 1141 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 1201 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 1261 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1321 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc 1381 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 1441 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 1501 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac 1561 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 1621 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 1681 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 1741 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 1801 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 1861 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 1921 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 1981 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 2041 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 2101 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 2161 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 2221 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 2281 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 2341 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 2401 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca 2461 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 2521 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc 2581 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc 2641 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat 2701 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac 2761 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggtcatca 2821 tcatcatcat catcacgatt atgatattcc tactactgag aatttgtatt ttcagggttc 2881 taccttcagt aaacttcgcg aacaactggg ccccgtgacg caggaattct gggacaacct 2941 ggaaaaagaa accgagggac tgcgtcagga aatgtccaaa gatttagaag aggtgaaggc 3001 caaggttcag ccatatctcg atgactttca gaaaaaatgg caggaagaga tggaattata 3061 tcgtcaaaag gtggaaccgc tgcgtgcgga actgcaagag ggggcacgcc aaaaactcca 3121 tgagctccaa gagaagctca gcccattagg cgaagaaatg cgcgatcgcg cccgtgcaca 3181 tgttgatgca ctccggactc atttggcgcc gtattcggat gaacttcgcc agcgtttggc 3241 cgcacgtctc gaggcgctga aagaaaacgg gggtgcccgc ttggctgagt accacgcgaa 3301 agcgacagaa cacctgagca ccttgagcga aaaagcgaaa ccggcgctgg aagatctacg 3361 ccagggctta ttgcctgttc ttgagagctt taaagtcagt tttctgtcag ctctggaaga 3421 atatactaaa aagctgaata cccagtaagc ttgcggccgc actcgagcac caccaccacc 3481 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg 3541 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 3601 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag 3661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 3841 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 3901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 3961 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 4021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 4081 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 4141 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag 4201 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag 4261 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg 4321 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc 4381 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg 4441 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc 4501 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa 4561 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa 4621 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa 4681 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa 4741 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc 4801 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg 4861 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt 4921 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc 4981 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat 5041 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5101 aaaagatcaa aggatcttct tgaaatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5161 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5221 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 5281 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5341 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5401 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5461 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5521 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5581 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5641 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 5701 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 5761 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 5821 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 5881 a //