LOCUS       sequence-385334-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(I130D).
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: pBEL2556
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(116..307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1116..2198)
                     /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     2160..2179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2199..2276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2482..2501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2585..2604
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2585..2603
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2604..2628
                     /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             2677..2694
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2695..2715
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3351..3369)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3365..3412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3449..3904
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3536..3555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3746..3767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3979..4012)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4091..4195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4196..4264,4265..5056)
                     /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(4414..4433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5227..5815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5716..5735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5969..5986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6001..6126,1..14)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6066..6085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
       61 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
      121 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
      181 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
      241 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
      301 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
      361 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
      421 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
      481 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
      541 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
      601 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
      661 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
      721 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
      781 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
      841 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
      901 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
      961 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1021 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1081 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1141 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1201 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     1261 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     1321 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     1381 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1441 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     1501 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1561 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1621 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1681 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1741 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1801 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1861 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1921 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1981 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2041 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     2101 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     2161 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     2221 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     2281 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     2341 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     2401 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     2461 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     2521 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     2581 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     2641 attttgttta actttggccc aggagatata catatgcatc accaccatca ccatgaaaat
     2701 ctgtattttc aggcagacca gaccaatccg tataagctga tggacgaggc ggcgcagaaa
     2761 acgttcgatc gcctgaagaa tgagcaaccg caaattcggg ccaacccgga ttatctgcgt
     2821 accattgttg atcaggaact gctgccatac gtacaggtga aatacgccgg tgcgctggtg
     2881 ctgggccagt attacaagag tgcgacccct gctcaacgtg aagcctactt tgccgctttc
     2941 cgtgagtacc tgaagcaggc ttacggtcag gcgctggcga tgtatcacgg tcaaacctat
     3001 cagattgcgc cagaacagcc gctgggcgat aaaaccgatg tgcctattcg cgttaccatt
     3061 attgacccga atggccgtcc gccggtgcgt ctggacttcc agtggcgtaa aaactcccag
     3121 acgggcaatt ggcaggctta cgacatgatt gctgaaggcg tcagtatgat caccaccaaa
     3181 caaaacgagt ggggaacgct gctgcgtacc aaaggtatcg acggcctgac tgcgcaactg
     3241 aaatcgattt ctcaacagaa aatcactctg gaagagaaaa aataagagag accggccggc
     3301 cgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3361 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3421 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     3481 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3541 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3601 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3661 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3721 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3781 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3841 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     3901 atttaggtgg cacttttcgg ggaacgacga cctgcagcca agctagcttg gctggacgta
     3961 aactcctctt cagacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg
     4021 ttattgaata tgatcggaat tgggctgcag gaattcgata gcttggctgc aggtcgacct
     4081 cgaggatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4141 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4201 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4261 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4321 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4381 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4441 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4501 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4561 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4621 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4681 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     4741 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4801 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4861 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gctctcgcgg
     4921 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4981 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5041 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5101 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5161 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5221 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5281 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5341 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5401 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5461 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5521 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5581 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5641 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5701 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5761 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5821 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5881 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5941 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6001 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     6061 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     6121 gtgact
//
