LOCUS       Exported                6079 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of an extended membrane scaffold protein 
            (MSP1E3D1) for Nanodisc formation.
ACCESSION   .
VERSION     .
KEYWORDS    pMSP1E3D1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6079)
  AUTHORS   Denisov IG, Baas BJ, Grinkova YV, Sligar SG
  TITLE     Cooperativity in cytochrome P450 3A4: linkages in substrate binding,
            spin state, uncoupling, and product formation.
  JOURNAL   J Biol Chem. 2007 Mar 9. 282(10):7066-76.
  PUBMED    17213193
REFERENCE   2  (bases 1 to 6079)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6079
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    5..144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(246..437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1246..2328)
                     /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     2290..2309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2329..2406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2715..2734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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             2773..2795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2808..2828
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             2850..2870
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3658..3675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3728..3746)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3742..3789
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3826..4281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3913..3932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4123..4144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4373..5188)
                     /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     5096..5115
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5180..5199
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5310..5898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5799..5818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6052..6069
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac catgggtcat catcatcatc
     2821 atcatcacga ttatgatatt cctactactg agaatttgta ttttcagggt tctaccttca
     2881 gtaaacttcg cgaacaactg ggccccgtga cgcaggaatt ctgggacaac ctggaaaaag
     2941 aaaccgaggg actgcgtcag gaaatgtcca aagatttaga agaggtgaag gccaaggttc
     3001 agccatatct cgatgacttt cagaaaaaat ggcaggaaga gatggaatta tatcgtcaaa
     3061 aggtggaacc gctgcgtgcg gaactgcaag agggggcacg ccaaaaactc catgagctcc
     3121 aagagaagct cagcccatta ggcgaagaaa tgcgcgatcg cgcccgtgca catgttgatg
     3181 cactccggac tcatttggcg ccatatctcg atgactttca gaaaaaatgg caggaagaga
     3241 tggaattata tcgtcaaaag gtggaaccgc tgcgtgcgga actgcaagag ggggcacgcc
     3301 aaaaactcca tgagctccaa gagaagctca gcccattagg cgaagaaatg cgcgatcgcg
     3361 cccgtgcaca tgttgatgca ctccggactc atttggcgcc gtattcggat gaacttcgcc
     3421 agcgtttggc cgcacgtctc gaggcgctga aagaaaacgg gggtgcccgc ttggctgagt
     3481 accacgcgaa agcgacagaa cacctgagca ccttgagcga aaaagcgaaa ccggcgctgg
     3541 aagatctacg ccagggctta ttgcctgttc ttgagagctt taaagtcagt tttctgtcag
     3601 ctctggaaga atatactaaa aagctgaata cccagtaagc ttgcggccgc actcgagcac
     3661 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     3721 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     3781 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     3841 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3901 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3961 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4021 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4081 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4141 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4201 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4261 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4321 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     4381 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     4441 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     4501 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     4561 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     4621 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     4681 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     4741 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     4801 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     4861 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     4921 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     4981 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     5041 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     5101 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     5161 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     5221 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5281 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5341 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5401 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     5461 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5521 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5581 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5641 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5701 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5761 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5821 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5881 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5941 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6001 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6061 gtgagcgagg aagcggaag
//
