LOCUS       sequence-384221-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(A168R).
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: pBEL2552
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     156..178
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(172..363)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1172..2254)
                     /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     2216..2235
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2255..2332)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2538..2557
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2641..2660
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2641..2659
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2660..2684
                     /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             2733..2750
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2751..2771
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3407..3425)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3421..3468
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3505..3960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3592..3611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3802..3823
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4035..4068)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4147..4251
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4252..4320,4321..5112)
                     /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(4470..4489)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5283..5871
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5772..5791
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6025..6042
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6057..6126,1..70)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(join(6122..6126,1..15))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
       61 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
      121 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
      181 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
      241 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
      301 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
      361 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
      421 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
      481 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
      541 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
      601 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
      661 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
      721 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
      781 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
      841 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
      901 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
      961 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     1021 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     1081 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     1141 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1201 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1261 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     1321 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     1381 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     1441 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     1501 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     1561 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     1621 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     1681 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     1741 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     1801 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     1861 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     1921 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     1981 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     2041 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     2101 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     2161 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     2221 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     2281 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     2341 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     2401 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     2461 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     2521 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     2581 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     2641 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     2701 tgtttaactt tggcccagga gatatacata tgcatcacca ccatcaccat gaaaatctgt
     2761 attttcaggc agaccagacc aatccgtata agctgatgga cgaggcggcg cagaaaacgt
     2821 tcgatcgcct gaagaatgag caaccgcaaa ttcgggccaa cccggattat ctgcgtacca
     2881 ttgttgatca ggaactgctg ccatacgtac aggtgaaata cgccggtgcg ctggtgctgg
     2941 gccagtatta caagagtgcg acccctgctc aacgtgaagc ctactttgcc gctttccgtg
     3001 agtacctgaa gcaggcttac ggtcaggcgc tggcgatgta tcacggtcaa acctatcaga
     3061 ttgcgccaga acagccgctg ggcgataaaa ccattgtgcc tattcgcgtt accattattg
     3121 acccgaatgg ccgtccgccg gtgcgtctgg acttccagtg gcgtaaaaac tcccagacgg
     3181 gcaattggca ggcttacgac atgattcgcg aaggcgtcag tatgatcacc accaaacaaa
     3241 acgagtgggg aacgctgctg cgtaccaaag gtatcgacgg cctgactgcg caactgaaat
     3301 cgatttctca acagaaaatc actctggaag agaaaaaata agagagaccg gccggccgat
     3361 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3421 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3481 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3541 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3601 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3661 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3721 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3781 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3841 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3901 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3961 aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg gacgtaaact
     4021 cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat taagggttat
     4081 tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt cgacctcgag
     4141 gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4201 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4261 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4321 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4381 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4441 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4501 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4561 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4621 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4681 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4741 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     4801 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4861 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4921 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc tcgcggtatc
     4981 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5041 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5101 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5161 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5221 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5281 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5341 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5401 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5461 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5521 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5581 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5641 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5701 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5761 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5821 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5881 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5941 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6001 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     6061 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     6121 gtacaa
//
