LOCUS       sequence-392553-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(V171R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  AUTHORS   MacRae MR, Puvanendran D, Haase MAB, Coudray N, Kolich L, Lam C, 
            Baek M, Bhabha G, Ekiert DC
  TITLE     Protein-protein interactions in the Mla lipid transport system 
            probed by computational structure prediction and deep mutational 
            scanning.
  JOURNAL   J Biol Chem. 2023 Apr 24:104744. doi: 10.1016/j.jbc.2023.104744.
  PUBMED    37100290
REFERENCE   2  (bases 1 to 6126)
  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: "J Biol 
            Chem. 2023 Apr 24:104744. doi: 10.1016/j.jbc.2023.104744."
COMMENT     Sequence Label: pBEL2572
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..34)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     175..197
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(191..382)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1191..2273)
                     /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     2235..2254
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2274..2351)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2557..2576
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2660..2679
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2660..2678
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2679..2703
                     /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             2752..2769
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2770..2790
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3426..3444)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3440..3487
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3524..3979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3611..3630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3821..3842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4054..4087)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4166..4270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4271..4339,4340..5131)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4489..4508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5302..5890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5791..5810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6044..6061
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6076..6126,1..89)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
       61 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
      121 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
      181 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
      241 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
      301 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
      361 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
      421 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
      481 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
      541 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
      601 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
      661 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
      721 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
      781 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
      841 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
      901 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
      961 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     1021 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     1081 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     1141 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     1201 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1261 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     1321 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     1381 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     1441 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     1501 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     1561 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     1621 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     1681 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     1741 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     1801 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     1861 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     1921 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     1981 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     2041 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     2101 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     2161 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     2221 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     2281 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     2341 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     2401 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     2461 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     2521 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     2581 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     2641 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     2701 tcccctctag aaataatttt gtttaacttt ggcccaggag atatacatat gcatcaccac
     2761 catcaccatg aaaatctgta ttttcaggca gaccagacca atccgtataa gctgatggac
     2821 gaggcggcgc agaaaacgtt cgatcgcctg aagaatgagc aaccgcaaat tcgggccaac
     2881 ccggattatc tgcgtaccat tgttgatcag gaactgctgc catacgtaca ggtgaaatac
     2941 gccggtgcgc tggtgctggg ccagtattac aagagtgcga cccctgctca acgtgaagcc
     3001 tactttgccg ctttccgtga gtacctgaag caggcttacg gtcaggcgct ggcgatgtat
     3061 cacggtcaaa cctatcagat tgcgccagaa cagccgctgg gcgataaaac cattgtgcct
     3121 attcgcgtta ccattattga cccgaatggc cgtccgccgg tgcgtctgga cttccagtgg
     3181 cgtaaaaact cccagacggg caattggcag gcttacgaca tgattgctga aggccgcagt
     3241 atgatcacca ccaaacaaaa cgagtgggga acgctgctgc gtaccaaagg tatcgacggc
     3301 ctgactgcgc aactgaaatc gatttctcaa cagaaaatca ctctggaaga gaaaaaataa
     3361 gagagaccgg ccggccgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3421 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3481 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     3541 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3601 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3661 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3721 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3781 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3841 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3901 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3961 atattaacgc ttacaattta ggtggcactt ttcggggaac gacgacctgc agccaagcta
     4021 gcttggctgg acgtaaactc ctcttcagac ctaataactt cgtatagcat acattatacg
     4081 aagttatatt aagggttatt gaatatgatc ggaattgggc tgcaggaatt cgatagcttg
     4141 gctgcaggtc gacctcgagg atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4201 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4261 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4321 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4381 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4441 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4501 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4561 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4621 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4681 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4741 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4801 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4861 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4921 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4981 gcgtggctct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5041 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5101 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5161 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5221 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5941 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6001 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6061 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     6121 ccgcaa
//
