LOCUS       sequence-309767-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant L246N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
  AUTHORS   Isom GL, Coudray N, MacRae MR, McManus CT, Ekiert DC, Bhabha G
  TITLE     LetB Structure Reveals a Tunnel for Lipid Transport across the 
            Bacterial Envelope.
  JOURNAL   Cell. 2020 Apr 30;181(3):653-664.e19. doi: 
            10.1016/j.cell.2020.03.030.
  PUBMED    32359438
REFERENCE   2  (bases 1 to 8057)
  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"; doi: 
            "10.1016/j.cell.2020.03"; journalName: "Cell"; date: "2020-04-30- 
            30"; volume: "181"; issue: "3"; pages: "653-664.e19"
COMMENT     Sequence Label: pBEL1807
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..89
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     173..192
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        173..191
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    192..216
                     /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             2787..2804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2870..2888)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2884..2931
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2968..3423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3055..3074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3265..3286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3498..3531)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3610..3714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3715..3783,3784..4575)
                     /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(3933..3952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4746..5334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5235..5254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5488..5505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5520..5659
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5585..5604)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5745..5767
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5761..5952)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6761..7843)
                     /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     7805..7824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7844..7921)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
       61 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
      121 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
      181 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
      241 tttggcccag gcggccagga gggtctcaca tgcaggaccg gggtaatacc gtcaccatcg
      301 actttatgtc ggcggatggt attgttccag gccgtacgcc tgttcgttat cagggcgttg
      361 aagtcggaac agtgcaggat atcagcctca gcgacgatct tcgtaagatt gaagtcaagg
      421 tcagcatcaa gtccgatatg aaagatgcgc tgcgcgaaga gactcagttc tggctggtga
      481 cgccaaaagc atcgttggca ggtgtctccg ggctggacgc cctcgtcggt gggaactata
      541 tcggcatgat gccgggtaaa ggtaaagagc aggatcactt tgtcgcactc gatacccaac
      601 cgaaatatcg gctggacaat ggcgatctga tgatccacct gcaagccccc gatctcggtt
      661 cgctgaacag cggttcattg gtctatttcc gcaagatccc ggtgggaaaa gtctacgact
      721 atgccatcaa tcccaacaag caaggcgtgg tgattgatgt cctgatcgag cggcgtttta
      781 ccgatctggt gaaaaaaggt agccgtttct ggaacgtttc cggcgttgat gccaacgtca
      841 gtatcagtgg cgcgaaggtg aaactggaaa gcctggcggc aaacgttaac ggtgcgattg
      901 ccttcgattc accagaagag tcgaaacctg ccgaggcgga agataccttt ggtctgtatg
      961 aagatctggc ccacagccag cgtggcgtaa taataaaact ggaactgccg agtggggccg
     1021 gattaaccgc cgactcgacg ccgttaatgt atcaggggct ggaagtcgga cagctgacta
     1081 aactggattt aaatcctggt ggtaaagtca caggggaaat gaccgttgat cccagcgtcg
     1141 ttaccctgct tcgtgaaaat acccgcatcg aattacgcaa cccgaaatta tcccttagcg
     1201 atgctaatct cagcgccctg ctgaccggca aaaccttcga gctggtgccc ggcgatggcg
     1261 agccacgcaa agagttcgtt gttgtgccag gcgaaaaagc actgctgcat gaacctgatg
     1321 ttctgacgct gaccctgacc gcgccggaaa gttacggtat tgatgcgggt cagccgctca
     1381 ttcttcacgg cgtgcaggta ggccaggtta tcgatcgtaa actcaccagc aaaggcgtca
     1441 cctttaccgt cgccatcgag cctcagcatc gcgaactggt aaaaggcgat agcaaatttg
     1501 tcgtcaacag ccgtgtcgat gtgaaggtgg ggctggatgg cgttgagttt ctcggtgcca
     1561 gcgcctcaga atggatcaat ggcgggatac gtattctgcc gggcgataaa ggtgagatga
     1621 aagccagcta tccactgtat gccaatttgg aaaaagcgct ggagaacagc cttagcgatt
     1681 tacccaccac aaccgtgagt ttgagtgcag agacgctgcc ggatgtgcag gcaggatcgg
     1741 tagtgctgta ccgtaaattt gaagttggtg aagtgattac cgtgcgtccg cgagctaacg
     1801 cgtttgatat cgatctgcat attaagccgg agtatcgcaa ccttctgacc agcaatagcg
     1861 tgttctgggc agaaggcggg gcgaaagttc agctgaatgg tagtggtctg accgtacagg
     1921 catccccgct ctccagagca ttaaagggag ccattagctt cgacaacctc agcggtgcca
     1981 gcgccagtca gcgtaaaggc gataagcgta ttctgtatgc ttccgaaaca gcggcccgtg
     2041 cggttggcgg gcagattacg cttcacgctt tcgatgccgg aaaactggcg gtcgggatgc
     2101 caattcgcta tctcggtatt gatatcgggc aaatccagac gctggatctg attaccgcac
     2161 gcaatgaagt acaggcaaag gcggtacttt acccggaata tgtccagacc tttgctcgcg
     2221 gtggtacgcg cttctcagtg gtcacaccgc aaatttcggc agctggcgtt gagcatcttg
     2281 atactatcct ccagccgtat atcaacgtcg aaccaggccg gggcaatcct cgccgcgact
     2341 ttgaattaca agaggccacc attactgatt cgcgttacct ggatggctta agcattattg
     2401 ttgaagcgcc ggaagccggt tcgttaggca tcggtacgcc tgtgctgttc cgtggtctgg
     2461 aagtcggtac ggttacagga atgacgctgg ggacattgtc agatcgcgtg atgattgcga
     2521 tgcgcatcag taaacgctat caacacctgg tgcgtaacaa ttccgtcttc tggttggcat
     2581 cgggttacag tctggacttt ggtctgacgg gcggcgtagt gaaaaccggc acctttaacc
     2641 agtttatccg tggcggcatc gccttcgcca cgcctccggg tacgccactg gcaccgaaag
     2701 cccaggaagg caaacacttc ctgttgcagg aaagtgaacc gaaagagtgg cgtgaatggg
     2761 gaactgcgct tcccaaaggc gagacccatc accaccatca ccattaaggc caggccggcc
     2821 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2881 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2941 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     3001 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3061 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3121 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3181 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3241 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3301 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3361 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     3421 tttaggtggc acttttcggg gaacgacgac ctgcagccaa gctagcttgg ctggacgtaa
     3481 actcctcttc agacctaata acttcgtata gcatacatta tacgaagtta tattaagggt
     3541 tattgaatat gatcggaatt gggctgcagg aattcgatag cttggctgca ggtcgacctc
     3601 gaggatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     3961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     4261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ctctcgcggt
     4441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5521 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5581 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5641 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5701 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5761 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5821 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5881 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5941 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     6001 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     6061 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     6121 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     6181 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     6241 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     6301 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     6361 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     6421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     6481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     6541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     6601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     6661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     6721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     6901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     6961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     7021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     7081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     7141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     7201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     7261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     7321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     7381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     7441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     7501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     7561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     7621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     7681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     7741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     7801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     7861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     7921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     7981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     8041 agtcccccgg ccacggg
//
