LOCUS       sequence-384569-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(D165I).
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: pBEL2553
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..128
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(122..313)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1122..2204)
                     /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     2166..2185
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2205..2282)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2488..2507
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2591..2610
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2591..2609
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2610..2634
                     /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             2683..2700
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2701..2721
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3357..3375)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3371..3418
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3455..3910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3542..3561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3752..3773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3985..4018)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4097..4201
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4202..4270,4271..5062)
                     /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(4420..4439)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5233..5821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5722..5741
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5975..5992
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6007..6126,1..20)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6072..6091)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
       61 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      121 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
      181 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
      241 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
      301 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
      361 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
      421 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      481 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      541 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      601 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      661 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      721 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      781 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
      841 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
      901 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
      961 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1021 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1081 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     1141 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1201 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1261 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1321 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     1381 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1441 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1501 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1561 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1621 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     1681 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     1741 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     1801 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     1861 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     1921 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     1981 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2041 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2101 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2161 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2221 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     2281 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     2341 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     2401 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     2461 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     2521 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     2581 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2641 gaaataattt tgtttaactt tggcccagga gatatacata tgcatcacca ccatcaccat
     2701 gaaaatctgt attttcaggc agaccagacc aatccgtata agctgatgga cgaggcggcg
     2761 cagaaaacgt tcgatcgcct gaagaatgag caaccgcaaa ttcgggccaa cccggattat
     2821 ctgcgtacca ttgttgatca ggaactgctg ccatacgtac aggtgaaata cgccggtgcg
     2881 ctggtgctgg gccagtatta caagagtgcg acccctgctc aacgtgaagc ctactttgcc
     2941 gctttccgtg agtacctgaa gcaggcttac ggtcaggcgc tggcgatgta tcacggtcaa
     3001 acctatcaga ttgcgccaga acagccgctg ggcgataaaa ccattgtgcc tattcgcgtt
     3061 accattattg acccgaatgg ccgtccgccg gtgcgtctgg acttccagtg gcgtaaaaac
     3121 tcccagacgg gcaattggca ggcttacatt atgattgctg aaggcgtcag tatgatcacc
     3181 accaaacaaa acgagtgggg aacgctgctg cgtaccaaag gtatcgacgg cctgactgcg
     3241 caactgaaat cgatttctca acagaaaatc actctggaag agaaaaaata agagagaccg
     3301 gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     3361 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3421 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3481 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3541 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3601 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3661 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3721 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3781 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3841 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3901 cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg
     3961 gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat
     4021 taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt
     4081 cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4141 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4201 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4261 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4321 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4381 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4441 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4501 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4561 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4621 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4681 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4741 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4801 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4861 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc
     4921 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4981 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5041 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5101 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5161 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5221 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5281 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5341 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5401 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5461 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5521 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5581 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5641 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5701 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5761 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5821 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5881 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5941 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6001 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     6061 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     6121 cgctac
//
