LOCUS       sequence-382984-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(Y72A).
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: pBEL2573
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..23)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     164..186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(180..371)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1180..2262)
                     /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     2224..2243
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2263..2340)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2546..2565
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2649..2668
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2649..2667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2668..2692
                     /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             2741..2758
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2759..2779
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3415..3433)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3429..3476
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3513..3968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3600..3619)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3810..3831
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4043..4076)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4155..4259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4260..4328,4329..5120)
                     /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(4478..4497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5291..5879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5780..5799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6033..6050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6065..6126,1..78)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
       61 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
      121 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
      181 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
      241 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
      301 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
      361 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
      421 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
      481 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
      541 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
      601 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
      661 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
      721 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
      781 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
      841 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
      901 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
      961 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     1021 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     1081 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     1141 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1201 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1261 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     1321 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     1381 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     1441 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     1501 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     1561 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     1621 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     1681 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     1741 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     1801 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     1861 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     1921 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     1981 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     2041 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     2101 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     2161 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     2221 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     2281 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     2341 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     2401 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     2461 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     2521 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     2581 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     2641 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     2701 aataattttg tttaactttg gcccaggaga tatacatatg catcaccacc atcaccatga
     2761 aaatctgtat tttcaggcag accagaccaa tccgtataag ctgatggacg aggcggcgca
     2821 gaaaacgttc gatcgcctga agaatgagca accgcaaatt cgggccaacc cggattatct
     2881 gcgtaccatt gttgatcagg aactgctgcc atacgtacag gtgaaagcgg ccggtgcgct
     2941 ggtgctgggc cagtattaca agagtgcgac ccctgctcaa cgtgaagcct actttgccgc
     3001 tttccgtgag tacctgaagc aggcttacgg tcaggcgctg gcgatgtatc acggtcaaac
     3061 ctatcagatt gcgccagaac agccgctggg cgataaaacc attgtgccta ttcgcgttac
     3121 cattattgac ccgaatggcc gtccgccggt gcgtctggac ttccagtggc gtaaaaactc
     3181 ccagacgggc aattggcagg cttacgacat gattgctgaa ggcgtcagta tgatcaccac
     3241 caaacaaaac gagtggggaa cgctgctgcg taccaaaggt atcgacggcc tgactgcgca
     3301 actgaaatcg atttctcaac agaaaatcac tctggaagag aaaaaataag agagaccggc
     3361 cggccgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     3421 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     3481 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     3541 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3601 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3661 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3721 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3781 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3841 caaccctatc tcggtctatt cttttgattt ataatggatt ttgccgattt cggcctattg
     3901 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3961 tacaatttag gtggcacttt tcggggaacg acgacctgca gccaagctag cttggctgga
     4021 cgtaaactcc tcttcagacc taataacttc gtatagcata cattatacga agttatatta
     4081 agggttattg aatatgatcg gaattgggct gcaggaattc gatagcttgg ctgcaggtcg
     4141 acctcgagga tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4201 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4261 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4321 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4381 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4441 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4501 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4561 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4621 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4681 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4741 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4801 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4861 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4921 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggctctc
     4981 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5041 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5101 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5161 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5221 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5281 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5341 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5401 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     5461 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5521 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5581 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5641 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5701 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5761 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5821 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5881 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5941 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6001 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6061 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     6121 cactct
//
