LOCUS       sequence-383649-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(R134E).
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: pBEL2563
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..54)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     195..217
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(211..402)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1211..2293)
                     /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     2255..2274
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2294..2371)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2577..2596
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2680..2699
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2680..2698
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2699..2723
                     /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             2772..2789
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2790..2810
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3446..3464)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3460..3507
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3544..3999
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3631..3650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3841..3862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4074..4107)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4186..4290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4291..4359,4360..5151)
                     /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(4509..4528)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5322..5910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5811..5830
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6064..6081
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6096..6126,1..109)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
       61 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
      121 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
      181 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
      241 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
      301 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
      361 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
      421 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
      481 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
      541 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
      601 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
      661 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
      721 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
      781 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
      841 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
      901 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
      961 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     1021 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     1081 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     1141 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     1201 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1261 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     1321 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     1381 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1441 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1501 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1561 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1621 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1681 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1741 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     1801 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     1861 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     1921 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     1981 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     2041 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     2101 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     2161 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     2221 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     2281 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     2341 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     2401 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     2461 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     2521 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     2581 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     2641 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     2701 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt ggcccaggag
     2761 atatacatat gcatcaccac catcaccatg aaaatctgta ttttcaggca gaccagacca
     2821 atccgtataa gctgatggac gaggcggcgc agaaaacgtt cgatcgcctg aagaatgagc
     2881 aaccgcaaat tcgggccaac ccggattatc tgcgtaccat tgttgatcag gaactgctgc
     2941 catacgtaca ggtgaaatac gccggtgcgc tggtgctggg ccagtattac aagagtgcga
     3001 cccctgctca acgtgaagcc tactttgccg ctttccgtga gtacctgaag caggcttacg
     3061 gtcaggcgct ggcgatgtat cacggtcaaa cctatcagat tgcgccagaa cagccgctgg
     3121 gcgataaaac cattgtgcct attgaagtta ccattattga cccgaatggc cgtccgccgg
     3181 tgcgtctgga cttccagtgg cgtaaaaact cccagacggg caattggcag gcttacgaca
     3241 tgattgctga aggcgtcagt atgatcacca ccaaacaaaa cgagtgggga acgctgctgc
     3301 gtaccaaagg tatcgacggc ctgactgcgc aactgaaatc gatttctcaa cagaaaatca
     3361 ctctggaaga gaaaaaataa gagagaccgg ccggccgatc cggctgctaa caaagcccga
     3421 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3481 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     3541 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3601 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3661 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3721 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3781 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3841 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3901 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3961 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaac
     4021 gacgacctgc agccaagcta gcttggctgg acgtaaactc ctcttcagac ctaataactt
     4081 cgtatagcat acattatacg aagttatatt aagggttatt gaatatgatc ggaattgggc
     4141 tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg aacccctatt
     4201 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4261 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4321 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4381 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4441 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4501 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4561 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4621 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4681 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4741 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4801 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     4861 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4921 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4981 gataaatctg gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat
     5041 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5101 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5161 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5221 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5281 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5341 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5401 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5461 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5521 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5581 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5641 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5701 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5761 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5821 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5881 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5941 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6001 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6061 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     6121 catctg
//
