LOCUS       sequence-383654-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(R186A).
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: pBEL2566
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    14..153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(79..98)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     239..261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(255..446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1255..2337)
                     /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     2299..2318
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2338..2415)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2621..2640
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2724..2743
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2724..2742
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2743..2767
                     /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             2816..2833
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2834..2854
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3490..3508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3504..3551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3588..4043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3675..3694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3885..3906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4118..4151)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4230..4334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4335..4403,4404..5195)
                     /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(4553..4572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5366..5954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5855..5874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6108..6125
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
       61 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      121 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
      181 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
      241 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
      301 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
      361 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
      421 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
      481 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
      541 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
      601 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
      661 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
      721 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
      781 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
      841 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
      901 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
      961 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     1021 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     1081 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     1141 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     1201 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     1261 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1321 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     1381 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     1441 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     1501 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     1561 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     1621 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     1681 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     1741 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     1801 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     1861 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     1921 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     1981 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     2041 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     2101 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     2161 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     2221 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     2281 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     2341 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     2401 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     2461 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     2521 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     2581 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     2641 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     2701 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     2761 caattcccct ctagaaataa ttttgtttaa ctttggccca ggagatatac atatgcatca
     2821 ccaccatcac catgaaaatc tgtattttca ggcagaccag accaatccgt ataagctgat
     2881 ggacgaggcg gcgcagaaaa cgttcgatcg cctgaagaat gagcaaccgc aaattcgggc
     2941 caacccggat tatctgcgta ccattgttga tcaggaactg ctgccatacg tacaggtgaa
     3001 atacgccggt gcgctggtgc tgggccagta ttacaagagt gcgacccctg ctcaacgtga
     3061 agcctacttt gccgctttcc gtgagtacct gaagcaggct tacggtcagg cgctggcgat
     3121 gtatcacggt caaacctatc agattgcgcc agaacagccg ctgggcgata aaaccattgt
     3181 gcctattcgc gttaccatta ttgacccgaa tggccgtccg ccggtgcgtc tggacttcca
     3241 gtggcgtaaa aactcccaga cgggcaattg gcaggcttac gacatgattg ctgaaggcgt
     3301 cagtatgatc accaccaaac aaaacgagtg gggaacgctg ctggcgacca aaggtatcga
     3361 cggcctgact gcgcaactga aatcgatttc tcaacagaaa atcactctgg aagagaaaaa
     3421 ataagagaga ccggccggcc gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3481 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3541 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3601 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3661 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3721 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3781 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3841 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3901 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3961 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4021 caaaatatta acgcttacaa tttaggtggc acttttcggg gaacgacgac ctgcagccaa
     4081 gctagcttgg ctggacgtaa actcctcttc agacctaata acttcgtata gcatacatta
     4141 tacgaagtta tattaagggt tattgaatat gatcggaatt gggctgcagg aattcgatag
     4201 cttggctgca ggtcgacctc gaggatgtgc gcggaacccc tatttgttta tttttctaaa
     4261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4861 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     4921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5041 gtgagcgtgg ctctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6121 gcgagg
//
