LOCUS       sequence-385336-        6012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaD(L123A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6012)
  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 6012)
  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: pBEL1910
FEATURES             Location/Qualifiers
     source          1..6012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    21..160
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(86..105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     246..268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(262..453)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1262..2344)
                     /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     2306..2325
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2345..2422)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2628..2647
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2731..2750
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2731..2749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2750..2774
                     /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             2823..2840
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3383..3401)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3397..3444
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3481..3936
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3568..3587)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3778..3799
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4011..4044)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4123..4227
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4228..4296,4297..5088)
                     /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(4446..4465)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5259..5847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5748..5767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6001..6012,1..6)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
       61 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      121 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
      181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
      241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      301 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      361 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      421 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      481 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      541 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      601 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
      661 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
      721 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
      781 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
      841 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
      901 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
      961 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     1021 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     1081 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     1141 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     1201 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     1261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1321 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     1381 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     1441 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     1501 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     1561 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1621 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1681 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1741 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1801 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1861 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1921 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1981 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     2041 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     2101 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     2161 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     2221 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     2281 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     2341 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     2401 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     2461 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     2521 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     2581 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     2641 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     2701 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     2761 cggataacaa ttcccctcta gaaataattt tgtttaactt tggcccagga gatatacata
     2821 tgcatcacca ccatcaccat gaaaatctgt attttcagac gtccatacgt actgaaccga
     2881 cctacacgct ttatgcgacg ttcgataaca ttggcggcct gaaagcgcgc tctccggtca
     2941 gtattggtgg cgttgttgtg ggtcgggtgg cggatattac gctggacccg aaaacctatc
     3001 tgccgcgcgt aacgctggaa attgaacaac gttataacca cattccagat accagttcgc
     3061 tgagcattcg tacttccggc ctgctggggg aacaatatct ggcattaaac gtcggttttg
     3121 aagacccgga agcggggact gctatcctga aggatggcga tacaattcag gacactaagt
     3181 ctgcgatggt gctggaagat ctcattggtc agttccttta cggtagtaaa ggcgatgaca
     3241 ataagaatag tggcgatgcg ccagctgctg cgccaggtaa taatgaaacc actgaacctg
     3301 tgggtacaac gaaataagag agaccggccg gccgatccgg ctgctaacaa agcccgaaag
     3361 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3421 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     3481 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3541 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3601 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3661 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3721 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3781 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3841 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3901 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaacgac
     3961 gacctgcagc caagctagct tggctggacg taaactcctc ttcagaccta ataacttcgt
     4021 atagcataca ttatacgaag ttatattaag ggttattgaa tatgatcgga attgggctgc
     4081 aggaattcga tagcttggct gcaggtcgac ctcgaggatg tgcgcggaac ccctatttgt
     4141 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4201 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4261 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4321 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4381 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4441 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4501 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4561 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4621 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4681 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4741 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4801 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4861 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4921 aaatctggag ccggtgagcg tggctctcgc ggtatcattg cagcactggg gccagatggt
     4981 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5041 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5101 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5161 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5221 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5281 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5341 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5401 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5461 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5521 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5581 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5641 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5701 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5761 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5821 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5881 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5941 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6001 agcgagtcag tg
//
