LOCUS       sequence-385349-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(G170K).
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: pBEL2555
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(29..220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1029..2111)
                     /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     2073..2092
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2112..2189)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2395..2414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2498..2517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2498..2516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2517..2541
                     /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             2590..2607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2608..2628
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3264..3282)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3278..3325
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3362..3817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3449..3468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3659..3680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3892..3925)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4004..4108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4109..4177,4178..4969)
                     /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(4327..4346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5140..5728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5629..5648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5882..5899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5914..6053
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5979..5998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
       61 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
      121 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
      181 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
      241 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
      301 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
      361 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
      421 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
      481 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
      541 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
      601 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
      661 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
      721 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
      781 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
      841 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
      901 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
      961 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     1021 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1081 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     1141 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     1201 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     1261 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     1321 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     1381 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     1441 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     1501 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     1561 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     1621 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     1681 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     1741 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     1801 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     1861 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     1921 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     1981 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     2041 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     2101 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     2161 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     2221 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     2281 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     2341 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     2401 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     2461 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     2521 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttgg cccaggagat
     2581 atacatatgc atcaccacca tcaccatgaa aatctgtatt ttcaggcaga ccagaccaat
     2641 ccgtataagc tgatggacga ggcggcgcag aaaacgttcg atcgcctgaa gaatgagcaa
     2701 ccgcaaattc gggccaaccc ggattatctg cgtaccattg ttgatcagga actgctgcca
     2761 tacgtacagg tgaaatacgc cggtgcgctg gtgctgggcc agtattacaa gagtgcgacc
     2821 cctgctcaac gtgaagccta ctttgccgct ttccgtgagt acctgaagca ggcttacggt
     2881 caggcgctgg cgatgtatca cggtcaaacc tatcagattg cgccagaaca gccgctgggc
     2941 gataaaacca ttgtgcctat tcgcgttacc attattgacc cgaatggccg tccgccggtg
     3001 cgtctggact tccagtggcg taaaaactcc cagacgggca attggcaggc ttacgacatg
     3061 attgctgaaa aagtcagtat gatcaccacc aaacaaaacg agtggggaac gctgctgcgt
     3121 accaaaggta tcgacggcct gactgcgcaa ctgaaatcga tttctcaaca gaaaatcact
     3181 ctggaagaga aaaaataaga gagaccggcc ggccgatccg gctgctaaca aagcccgaaa
     3241 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3301 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     3361 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3421 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3481 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3541 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3601 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3661 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3721 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3781 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaacga
     3841 cgacctgcag ccaagctagc ttggctggac gtaaactcct cttcagacct aataacttcg
     3901 tatagcatac attatacgaa gttatattaa gggttattga atatgatcgg aattgggctg
     3961 caggaattcg atagcttggc tgcaggtcga cctcgaggat gtgcgcggaa cccctatttg
     4021 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4081 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4141 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4201 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4261 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4321 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4381 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4441 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4501 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4561 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4621 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     4681 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4741 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4801 taaatctgga gccggtgagc gtggctctcg cggtatcatt gcagcactgg ggccagatgg
     4861 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4921 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4981 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5041 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5101 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5161 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5221 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5281 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5341 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5401 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5461 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5521 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5581 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5641 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5701 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5761 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5821 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5881 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5941 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     6001 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     6061 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     6121 gacaag
//
