LOCUS       sequence-383651-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(R147D).
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: pBEL2564
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..25)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     166..188
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(182..373)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1182..2264)
                     /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     2226..2245
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2265..2342)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2548..2567
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2651..2670
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2651..2669
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2670..2694
                     /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             2743..2760
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2761..2781
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3417..3435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3431..3478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3515..3970
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3602..3621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3812..3833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4045..4078)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4157..4261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4262..4330,4331..5122)
                     /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(4480..4499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5293..5881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5782..5801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6035..6052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6067..6126,1..80)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
       61 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      121 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      181 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
      241 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
      301 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
      361 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
      421 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
      481 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      541 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      601 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      661 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      721 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      781 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      841 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
      901 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
      961 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     1021 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1081 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1141 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     1201 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1261 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1321 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1381 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     1441 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1501 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1561 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1621 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1681 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     1741 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     1801 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     1861 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     1921 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     1981 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2041 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2101 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2161 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2221 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2281 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     2341 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     2401 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     2461 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     2521 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     2581 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     2641 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2701 gaaataattt tgtttaactt tggcccagga gatatacata tgcatcacca ccatcaccat
     2761 gaaaatctgt attttcaggc agaccagacc aatccgtata agctgatgga cgaggcggcg
     2821 cagaaaacgt tcgatcgcct gaagaatgag caaccgcaaa ttcgggccaa cccggattat
     2881 ctgcgtacca ttgttgatca ggaactgctg ccatacgtac aggtgaaata cgccggtgcg
     2941 ctggtgctgg gccagtatta caagagtgcg acccctgctc aacgtgaagc ctactttgcc
     3001 gctttccgtg agtacctgaa gcaggcttac ggtcaggcgc tggcgatgta tcacggtcaa
     3061 acctatcaga ttgcgccaga acagccgctg ggcgataaaa ccattgtgcc tattcgcgtt
     3121 accattattg acccgaatgg ccgtccgccg gtggatctgg acttccagtg gcgtaaaaac
     3181 tcccagacgg gcaattggca ggcttacgac atgattgctg aaggcgtcag tatgatcacc
     3241 accaaacaaa acgagtgggg aacgctgctg cgtaccaaag gtatcgacgg cctgactgcg
     3301 caactgaaat cgatttctca acagaaaatc actctggaag agaaaaaata agagagaccg
     3361 gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     3421 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3481 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3541 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3601 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3661 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3721 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3781 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3841 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3901 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3961 cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg
     4021 gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat
     4081 taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt
     4141 cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4201 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4261 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4321 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4381 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4441 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4501 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4561 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4621 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4681 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4741 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4801 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4861 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4921 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc
     4981 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5041 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5101 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5161 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5221 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5281 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5341 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5401 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5461 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5521 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5581 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5641 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5701 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5761 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5821 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5881 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5941 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6001 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6061 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     6121 tgcact
//
