LOCUS       sequence-383656-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(Y82K).
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: pBEL2574
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    6..145
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(71..90)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     231..253
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(247..438)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1247..2329)
                     /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     2291..2310
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2330..2407)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2613..2632
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2716..2735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2716..2734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2735..2759
                     /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             2808..2825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2826..2846
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3482..3500)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3496..3543
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3580..4035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3667..3686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3877..3898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4110..4143)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4222..4326
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4327..4395,4396..5187)
                     /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(4545..4564)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5358..5946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5847..5866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6100..6117
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
       61 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      121 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      181 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      241 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      301 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      361 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      421 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      481 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      541 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      601 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      661 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
      721 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      781 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
      841 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
      901 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
      961 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1021 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1081 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     1141 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     1201 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     1261 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1321 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     1381 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     1441 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     1501 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     1561 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     1621 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     1681 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     1741 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     1801 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     1861 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     1921 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     1981 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2041 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     2101 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     2161 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     2221 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     2281 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     2341 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     2401 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     2461 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     2521 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     2581 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     2641 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     2701 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     2761 ctctagaaat aattttgttt aactttggcc caggagatat acatatgcat caccaccatc
     2821 accatgaaaa tctgtatttt caggcagacc agaccaatcc gtataagctg atggacgagg
     2881 cggcgcagaa aacgttcgat cgcctgaaga atgagcaacc gcaaattcgg gccaacccgg
     2941 attatctgcg taccattgtt gatcaggaac tgctgccata cgtacaggtg aaatacgccg
     3001 gtgcgctggt gctgggccag tataaaaaga gtgcgacccc tgctcaacgt gaagcctact
     3061 ttgccgcttt ccgtgagtac ctgaagcagg cttacggtca ggcgctggcg atgtatcacg
     3121 gtcaaaccta tcagattgcg ccagaacagc cgctgggcga taaaaccatt gtgcctattc
     3181 gcgttaccat tattgacccg aatggccgtc cgccggtgcg tctggacttc cagtggcgta
     3241 aaaactccca gacgggcaat tggcaggctt acgacatgat tgctgaaggc gtcagtatga
     3301 tcaccaccaa acaaaacgag tggggaacgc tgctgcgtac caaaggtatc gacggcctga
     3361 ctgcgcaact gaaatcgatt tctcaacaga aaatcactct ggaagagaaa aaataagaga
     3421 gaccggccgg ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     3481 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     3541 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     3601 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3661 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3721 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3781 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3841 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3901 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3961 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4021 taacgcttac aatttaggtg gcacttttcg gggaacgacg acctgcagcc aagctagctt
     4081 ggctggacgt aaactcctct tcagacctaa taacttcgta tagcatacat tatacgaagt
     4141 tatattaagg gttattgaat atgatcggaa ttgggctgca ggaattcgat agcttggctg
     4201 caggtcgacc tcgaggatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4261 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4321 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4381 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4441 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4501 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4561 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4621 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4681 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4741 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4801 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4861 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4921 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4981 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5041 ggctctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     5101 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5161 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5221 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5281 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5341 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5401 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5461 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5521 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5581 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5641 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5701 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5761 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5821 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5881 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5941 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6001 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6061 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6121 gcggaa
//
