LOCUS       sequence-382995-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(N179E).
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: pBEL2561
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     155..177
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(171..362)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1171..2253)
                     /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     2215..2234
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2254..2331)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2537..2556
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2640..2659
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2640..2658
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2659..2683
                     /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             2732..2749
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2750..2770
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3406..3424)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3420..3467
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3504..3959
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3591..3610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3801..3822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4034..4067)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4146..4250
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4251..4319,4320..5111)
                     /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(4469..4488)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5282..5870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5771..5790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6024..6041
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6056..6126,1..69)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(join(6121..6126,1..14))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
       61 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
      121 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
      181 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
      241 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
      301 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
      361 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
      421 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
      481 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
      541 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
      601 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
      661 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
      721 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
      781 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
      841 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
      901 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
      961 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     1021 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     1081 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     1141 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1201 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1261 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     1321 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     1381 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     1441 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     1501 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     1561 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     1621 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     1681 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     1741 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     1801 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     1861 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     1921 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     1981 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     2041 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     2101 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     2161 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     2221 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     2281 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     2341 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     2401 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     2461 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     2521 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     2581 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     2641 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     2701 gtttaacttt ggcccaggag atatacatat gcatcaccac catcaccatg aaaatctgta
     2761 ttttcaggca gaccagacca atccgtataa gctgatggac gaggcggcgc agaaaacgtt
     2821 cgatcgcctg aagaatgagc aaccgcaaat tcgggccaac ccggattatc tgcgtaccat
     2881 tgttgatcag gaactgctgc catacgtaca ggtgaaatac gccggtgcgc tggtgctggg
     2941 ccagtattac aagagtgcga cccctgctca acgtgaagcc tactttgccg ctttccgtga
     3001 gtacctgaag caggcttacg gtcaggcgct ggcgatgtat cacggtcaaa cctatcagat
     3061 tgcgccagaa cagccgctgg gcgataaaac cattgtgcct attcgcgtta ccattattga
     3121 cccgaatggc cgtccgccgg tgcgtctgga cttccagtgg cgtaaaaact cccagacggg
     3181 caattggcag gcttacgaca tgattgctga aggcgtcagt atgatcacca ccaaacaaga
     3241 agagtgggga acgctgctgc gtaccaaagg tatcgacggc ctgactgcgc aactgaaatc
     3301 gatttctcaa cagaaaatca ctctggaaga gaaaaaataa gagagaccgg ccggccgatc
     3361 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     3421 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     3481 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3541 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3601 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3661 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3721 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3781 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3841 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3901 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     3961 ggtggcactt ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc
     4021 ctcttcagac ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt
     4081 gaatatgatc ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg
     4141 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4201 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4261 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4321 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4381 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4441 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4501 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4561 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4621 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4681 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4741 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     4801 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4861 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4921 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca
     4981 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5041 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5101 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5161 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5221 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5281 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5341 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5401 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     5461 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5521 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5581 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5641 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5701 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5761 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5821 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5881 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5941 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6001 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     6061 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     6121 tacaat
//
