LOCUS       sequence-382998-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression/purification of 6xHis-TEV-MlaC(E169R).
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: pBEL2554
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..46)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     187..209
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(203..394)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1203..2285)
                     /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     2247..2266
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2286..2363)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2569..2588
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2672..2691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2672..2690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2691..2715
                     /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             2764..2781
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2782..2802
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(3438..3456)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3452..3499
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3536..3991
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3623..3642)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3833..3854
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4066..4099)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4178..4282
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4283..4351,4352..5143)
                     /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(4501..4520)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5314..5902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5803..5822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6056..6073
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6088..6126,1..101)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
       61 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
      121 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
      181 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
      241 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
      301 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
      361 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
      421 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
      481 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
      541 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
      601 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
      661 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
      721 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
      781 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
      841 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
      901 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
      961 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     1021 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     1081 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     1141 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     1201 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1261 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     1321 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     1381 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     1441 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     1501 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     1561 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     1621 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     1681 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     1741 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     1801 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     1861 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     1921 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     1981 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     2041 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     2101 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     2161 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     2221 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     2281 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     2341 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     2401 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     2461 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     2521 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     2581 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     2641 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     2701 gcggataaca attcccctct agaaataatt ttgtttaact ttggcccagg agatatacat
     2761 atgcatcacc accatcacca tgaaaatctg tattttcagg cagaccagac caatccgtat
     2821 aagctgatgg acgaggcggc gcagaaaacg ttcgatcgcc tgaagaatga gcaaccgcaa
     2881 attcgggcca acccggatta tctgcgtacc attgttgatc aggaactgct gccatacgta
     2941 caggtgaaat acgccggtgc gctggtgctg ggccagtatt acaagagtgc gacccctgct
     3001 caacgtgaag cctactttgc cgctttccgt gagtacctga agcaggctta cggtcaggcg
     3061 ctggcgatgt atcacggtca aacctatcag attgcgccag aacagccgct gggcgataaa
     3121 accattgtgc ctattcgcgt taccattatt gacccgaatg gccgtccgcc ggtgcgtctg
     3181 gacttccagt ggcgtaaaaa ctcccagacg ggcaattggc aggcttacga catgattgct
     3241 cgcggcgtca gtatgatcac caccaaacaa aacgagtggg gaacgctgct gcgtaccaaa
     3301 ggtatcgacg gcctgactgc gcaactgaaa tcgatttctc aacagaaaat cactctggaa
     3361 gagaaaaaat aagagagacc ggccggccga tccggctgct aacaaagccc gaaaggaagc
     3421 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3481 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3541 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3601 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3661 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3721 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3781 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3841 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3901 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3961 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga acgacgacct
     4021 gcagccaagc tagcttggct ggacgtaaac tcctcttcag acctaataac ttcgtatagc
     4081 atacattata cgaagttata ttaagggtta ttgaatatga tcggaattgg gctgcaggaa
     4141 ttcgatagct tggctgcagg tcgacctcga ggatgtgcgc ggaaccccta tttgtttatt
     4201 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4261 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4321 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4381 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4441 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4501 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4561 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4621 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4681 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4741 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4801 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     4861 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4921 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4981 tggagccggt gagcgtggct ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5041 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5101 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5161 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5221 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5281 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5341 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5401 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5461 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5521 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5581 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5641 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5701 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5761 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5821 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5881 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5941 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6001 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6061 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     6121 cggtat
//
