LOCUS       sequence-309814-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant Y814Bpa.
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: pBEL1727
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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             4012..4029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4095..4113)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4109..4156
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4193..4648
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4280..4299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4490..4511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4723..4756)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4835..4939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4940..5008,5009..5800)
                     /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(5158..5177)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5971..6559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6460..6479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6713..6730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6745..6884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6810..6829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6970..6992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6986..7177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7986..8057,1..1011))
                     /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"
ORIGIN
        1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttgg cccaggcggc caggagggtc tcacatgcag
     1501 gaccggggta ataccgtcac catcgacttt atgtcggcgg atggtattgt tccaggccgt
     1561 acgcctgttc gttatcaggg cgttgaagtc ggaacagtgc aggatatcag cctcagcgac
     1621 gatcttcgta agattgaagt caaggtcagc atcaagtccg atatgaaaga tgcgctgcgc
     1681 gaagagactc agttctggct ggtgacgcca aaagcatcgt tggcaggtgt ctccgggctg
     1741 gacgccctcg tcggtgggaa ctatatcggc atgatgccgg gtaaaggtaa agagcaggat
     1801 cactttgtcg cactcgatac ccaaccgaaa tatcggctgg acaatggcga tctgatgatc
     1861 cacctgcaag cccccgatct cggttcgctg aacagcggtt cattggtcta tttccgcaag
     1921 atcccggtgg gaaaagtcta cgactatgcc atcaatccca acaagcaagg cgtggtgatt
     1981 gatgtcctga tcgagcggcg ttttaccgat ctggtgaaaa aaggtagccg tttctggaac
     2041 gtttccggcg ttgatgccaa cgtcagtatc agtggcgcga aggtgaaact ggaaagcctg
     2101 gcggcactgg ttaacggtgc gattgccttc gattcaccag aagagtcgaa acctgccgag
     2161 gcggaagata cctttggtct gtatgaagat ctggcccaca gccagcgtgg cgtaataata
     2221 aaactggaac tgccgagtgg ggccggatta accgccgact cgacgccgtt aatgtatcag
     2281 gggctggaag tcggacagct gactaaactg gatttaaatc ctggtggtaa agtcacaggg
     2341 gaaatgaccg ttgatcccag cgtcgttacc ctgcttcgtg aaaatacccg catcgaatta
     2401 cgcaacccga aattatccct tagcgatgct aatctcagcg ccctgctgac cggcaaaacc
     2461 ttcgagctgg tgcccggcga tggcgagcca cgcaaagagt tcgttgttgt gccaggcgaa
     2521 aaagcactgc tgcatgaacc tgatgttctg acgctgaccc tgaccgcgcc ggaaagttac
     2581 ggtattgatg cgggtcagcc gctcattctt cacggcgtgc aggtaggcca ggttatcgat
     2641 cgtaaactca ccagcaaagg cgtcaccttt accgtcgcca tcgagcctca gcatcgcgaa
     2701 ctggtaaaag gcgatagcaa atttgtcgtc aacagccgtg tcgatgtgaa ggtggggctg
     2761 gatggcgttg agtttctcgg tgccagcgcc tcagaatgga tcaatggcgg gatacgtatt
     2821 ctgccgggcg ataaaggtga gatgaaagcc agctatccac tgtatgccaa tttggaaaaa
     2881 gcgctggaga acagccttag cgatttaccc accacaaccg tgagtttgag tgcagagacg
     2941 ctgccggatg tgcaggcagg atcggtagtg ctgtaccgta aatttgaagt tggtgaagtg
     3001 attaccgtgc gtccgcgagc taacgcgttt gatatcgatc tgcatattaa gccggagtat
     3061 cgcaaccttc tgaccagcaa tagcgtgttc tgggcagaag gcggggcgaa agttcagctg
     3121 aatggtagtg gtctgaccgt acaggcatcc ccgctctcca gagcattaaa gggagccatt
     3181 agcttcgaca acctcagcgg tgccagcgcc agtcagcgta aaggcgataa gcgtattctg
     3241 tatgcttccg aaacagcggc ccgtgcggtt ggcgggcaga ttacgcttca cgctttcgat
     3301 gccggaaaac tggcggtcgg gatgccaatt cgctatctcg gtattgatat cgggcaaatc
     3361 cagacgctgg atctgattac cgcacgcaat gaagtacagg caaaggcggt actttacccg
     3421 gaatatgtcc agacctttgc tcgcggtggt acgcgcttct cagtggtcac accgcaaatt
     3481 tcggcagctg gcgttgagca tcttgatact atcctccagc cgtatatcaa cgtcgaacca
     3541 ggccggggca atcctcgccg cgactttgaa ttacaagagg ccaccattac tgattcgcgt
     3601 tacctggatg gcttaagcat tattgttgaa gcgccggaag ccggttcgtt aggcatcggt
     3661 acgcctgtgc tgttccgtgg tctggaagtc ggtacggtta caggaatgac gctggggaca
     3721 ttgtcagatc gcgtgatgat tgcgatgcgc atcagtaaac gctatcaaca cctggtgcgt
     3781 aacaattccg tcttctggtt ggcatcgggt tagagtctgg actttggtct gacgggcggc
     3841 gtagtgaaaa ccggcacctt taaccagttt atccgtggcg gcatcgcctt cgccacgcct
     3901 ccgggtacgc cactggcacc gaaagcccag gaaggcaaac acttcctgtt gcaggaaagt
     3961 gaaccgaaag agtggcgtga atggggaact gcgcttccca aaggcgagac ccatcaccac
     4021 catcaccatt aaggccaggc cggccgatcc ggctgctaac aaagcccgaa aggaagctga
     4081 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     4141 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     4201 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4261 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4321 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4381 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4441 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4501 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4561 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4621 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaacg acgacctgca
     4681 gccaagctag cttggctgga cgtaaactcc tcttcagacc taataacttc gtatagcata
     4741 cattatacga agttatatta agggttattg aatatgatcg gaattgggct gcaggaattc
     4801 gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga acccctattt gtttattttt
     4861 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4921 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4981 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5041 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5101 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5161 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5221 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5281 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5341 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5401 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5461 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5521 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5581 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5641 agccggtgag cgtggctctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5701 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5761 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5821 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5881 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5941 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6001 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6061 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     6121 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6181 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6241 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6301 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6361 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6421 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6481 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6541 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6601 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6661 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6721 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     6781 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6841 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     6901 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6961 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7021 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     7081 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     7141 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     7201 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     7261 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     7321 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     7381 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     7441 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     7501 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     7561 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     7621 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     7681 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     7741 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     7801 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     7861 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     7921 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     7981 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     8041 gccaacgcgc ggggaga
//
