LOCUS       sequence-375172-        5297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MsbA with a TEV protease cleavable N terminal His Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5297)
  AUTHORS   Lyu J, Liu C, Zhang T, Schrecke S, Elam NP, Packianathan C, Hochberg
            GKA, Russell D, Zhao M, Laganowsky A
  TITLE     Structural basis for lipid and copper regulation of the ABC 
            transporter MsbA.
  JOURNAL   Nat Commun. 2022 Nov 26;13(1):7291. doi: 10.1038/s41467-022-34905-2.
  PUBMED    36435815
REFERENCE   2  (bases 1 to 5297)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-11-26"; volume: "13(1):7291. doi"; pages: " 
            10.1038/s41467-022-34905-2"
COMMENT     Sequence Label: pCDF-His_TEV_MsbA
FEATURES             Location/Qualifiers
     source          1..5297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        88..106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    107..131
                     /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)."
     CDS             187..204
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             214..234
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     primer_bind     complement(2003..2021)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2017..2064
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2235..3026)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3027..3118)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(4179..5261)
                     /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     5223..5242
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(5262..5297,1..42))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
       61 ccttatgcga ctcctgcatt aggaaattaa tacgactcac tataggggaa ttgtgagcgg
      121 ataacaattc ccctgtagaa ataattttgt ttaactttaa taaggagata taccatgggc
      181 agcagccatc atcatcatca tcacagcagc ggcgaaaacc tgtattttca gatgcataac
      241 gacaaagatc tctctacgtg gcagacattc cgccgactgt ggccaaccat tgcgcctttc
      301 aaagcgggtc tgatcgtggc gggcgtagcg ttaatcctca acgcagccag cgataccttc
      361 atgttatcgc tccttaagcc acttcttgat gatggctttg gtaaaacaga tcgctccgtg
      421 ctggtgtgga tgccgctggt ggtgatcggg ctgatgattt tacgtggtat caccagctat
      481 gtctccagct actgtatctc ctgggtatca ggaaaggtgg taatgaccat gcgtcgccgc
      541 ctgtttggtc acatgatggg aatgccagtt tcattctttg acaaacagtc aacgggtacg
      601 ctgttgtcac gtattaccta cgattccgaa caggttgctt cttcttcttc cggcgcactg
      661 attactgttg tgcgtgaagg tgcgtcgatc atcggcctgt tcatcatgat gttctattac
      721 agttggcaac tgtcgatcat tttgattgtg ctggcaccga ttgtttcgat tgcgattcgc
      781 gttgtatcga agcgttttcg caacatcagt aaaaacatgc agaacaccat ggggcaggtg
      841 accaccagcg cagaacaaat gctgaagggc cacaaagaag tattgatttt cggtggtcag
      901 gaagtggaaa cgaaacgctt tgataaagtc agcaaccgaa tgcgtcttca ggggatgaaa
      961 atggtttcag cctcttccat ctctgatccg atcattcagc tgatcgcctc tttggcgctg
     1021 gcgtttgttc tgtatgcggc gagcttccca agtgtcatgg atagcctgac tgccggtacg
     1081 attaccgttg ttttctcttc aatgattgca ctgatgcgtc cgctgaaatc gctgaccaac
     1141 gttaacgccc agttccagcg cggtatggcg gcttgtcaga cgctgtttac cattctggac
     1201 agtgagcagg agaaagatga aggtaagcgc gtgatcgagc gtgcgactgg cgacgtggaa
     1261 ttccgcaatg tcacctttac ttatccggga cgtgacgtac ctgcattgcg taacatcaac
     1321 ctgaaaattc cggcagggaa gacggttgct ctggttggac gctctggttc gggtaaatca
     1381 accatcgcca gcctgatcac gcgtttttac gatattgatg aaggcgaaat cctgatggat
     1441 ggtcacgatc tgcgcgagta taccctggcg tcgttacgta accaggttgc tctggtgtcg
     1501 cagaatgtcc atctgtttaa cgatacggtt gctaacaaca ttgcttacgc acggactgaa
     1561 cagtacagcc gtgagcaaat tgaagaagcg gcgcgtatgg cctacgccat ggacttcatc
     1621 aataagatgg ataacggtct cgatacagtg attggtgaaa acggcgtgct gctctctggc
     1681 ggtcagcgtc agcgtattgc tatcgctcga gccttgttgc gtgatagccc gattctgatt
     1741 ctggacgaag ctacctcggc tctggatacc gaatccgaac gtgcgattca ggcggcactg
     1801 gatgagttgc agaaaaaccg tacctctctg gtgattgccc accgcttgtc taccattgaa
     1861 aaggcagacg aaatcgtggt cgtcgaggat ggtgtcattg tggaacgcgg tacgcataac
     1921 gatttgcttg agcaccgcgg cgtttacgcg caacttcaca aaatgcagtt tggccaataa
     1981 taattaacct aggctgctgc caccgctgag caataactag cataacccct tggggcctct
     2041 aaacgggtct tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca
     2101 ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga
     2161 ccctgccctg aaccgacgac cgggtcatcg tggccggatc ttgcggcccc tcggcttgaa
     2221 cgaattgtta gacattattt gccgactacc ttggtgatct cgcctttcac gtagtggaca
     2281 aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc
     2341 tgtctagctt caagtatgac gggctgatac tgggccggca ggcgctccat tgcccagtcg
     2401 gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat gcgggacaac
     2461 gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca tagcgttaag
     2521 gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc
     2581 gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat agccagatca
     2641 atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg ccattctcca
     2701 aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg cacaacaatg
     2761 gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt ttccaaaagg
     2821 tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt aaccagcaaa
     2881 tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa atgtacggcc
     2941 agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag ttgagtcgat
     3001 acttcggcga tcaccgcttc cctcatactc ttcctttttc aatattattg aagcatttat
     3061 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3121 gctagctcac tcggtcgcta cgctccgggc gtgagactgc ggcgggcgct gcggacacat
     3181 acaaagttac ccacagattc cgtggataag caggggacta acatgtgagg caaaacagca
     3241 gggccgcgcc ggtggcgttt ttccataggc tccgccctcc tgccagagtt cacataaaca
     3301 gacgcttttc cggtgcatct gtgggagccg tgaggctcaa ccatgaatct gacagtacgg
     3361 gcgaaacccg acaggactta aagatcccca ccgtttccgg cgggtcgctc cctcttgcgc
     3421 tctcctgttc cgaccctgcc gtttaccgga tacctgttcc gcctttctcc cttacgggaa
     3481 gtgtggcgct ttctcatagc tcacacactg gtatctcggc tcggtgtagg tcgttcgctc
     3541 caagctgggc tgtaagcaag aactccccgt tcagcccgac tgctgcgcct tatccggtaa
     3601 ctgttcactt gagtccaacc cggaaaagca cggtaaaacg ccactggcag cagccattgg
     3661 taactgggag ttcgcagagg atttgtttag ctaaacacgc ggttgctctt gaagtgtgcg
     3721 ccaaagtccg gctacactgg aaggacagat ttggttgctg tgctctgcga aagccagtta
     3781 ccacggttaa gcagttcccc aactgactta accttcgatc aaaccacctc cccaggtggt
     3841 tttttcgaag gttaagtcag ttggggaact gcttaacctt cgatcaaacc acctccccag
     3901 gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat
     3961 cctttgatct tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta
     4021 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     4081 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4141 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4201 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4261 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4321 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4381 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4441 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4501 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4561 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4621 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4681 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4741 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4801 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4861 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4921 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4981 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5041 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5101 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5161 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5221 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5281 ctctcttccg ggcgcta
//
