LOCUS       sequence-383655-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(T176R).
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: pBEL2568
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..35)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     176..198
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(192..383)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1192..2274)
                     /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     2236..2255
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2275..2352)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2558..2577
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2661..2680
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2661..2679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2680..2704
                     /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             2753..2770
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2771..2791
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3427..3445)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3441..3488
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3525..3980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3612..3631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3822..3843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4055..4088)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4167..4271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4272..4340,4341..5132)
                     /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(4490..4509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6045..6062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6077..6126,1..90)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
       61 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
      121 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
      181 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
      241 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
      301 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
      361 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
      421 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
      481 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
      541 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
      601 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
      661 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
      721 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
      781 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
      841 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
      901 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
      961 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1021 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1081 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1141 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1201 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1261 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1321 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1381 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1441 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1501 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1561 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1621 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     1681 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     1741 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     1801 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     1861 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     1921 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     1981 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2041 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2101 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2161 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2221 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2281 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2341 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     2401 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     2461 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     2521 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     2581 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     2641 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     2701 ttcccctcta gaaataattt tgtttaactt tggcccagga gatatacata tgcatcacca
     2761 ccatcaccat gaaaatctgt attttcaggc agaccagacc aatccgtata agctgatgga
     2821 cgaggcggcg cagaaaacgt tcgatcgcct gaagaatgag caaccgcaaa ttcgggccaa
     2881 cccggattat ctgcgtacca ttgttgatca ggaactgctg ccatacgtac aggtgaaata
     2941 cgccggtgcg ctggtgctgg gccagtatta caagagtgcg acccctgctc aacgtgaagc
     3001 ctactttgcc gctttccgtg agtacctgaa gcaggcttac ggtcaggcgc tggcgatgta
     3061 tcacggtcaa acctatcaga ttgcgccaga acagccgctg ggcgataaaa ccattgtgcc
     3121 tattcgcgtt accattattg acccgaatgg ccgtccgccg gtgcgtctgg acttccagtg
     3181 gcgtaaaaac tcccagacgg gcaattggca ggcttacgac atgattgctg aaggcgtcag
     3241 tatgatcacc cgcaaacaaa acgagtgggg aacgctgctg cgtaccaaag gtatcgacgg
     3301 cctgactgcg caactgaaat cgatttctca acagaaaatc actctggaag agaaaaaata
     3361 agagagaccg gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3421 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3481 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3541 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3601 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3661 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3721 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3781 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3841 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3901 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3961 aatattaacg cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct
     4021 agcttggctg gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac
     4081 gaagttatat taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt
     4141 ggctgcaggt cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4201 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4261 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4321 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4381 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4441 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4501 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4561 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4621 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4681 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4741 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4801 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4861 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4921 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4981 agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5041 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5101 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5161 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6001 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6061 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     6121 accgca
//
