LOCUS       sequence-309809-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant F468Bpa.
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: pBEL1725
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     966..985
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1005..1082)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1288..1307
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1391..1410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1391..1409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1410..1434
                     /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             4005..4022
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4088..4106)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4102..4149
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4186..4641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4273..4292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4483..4504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4716..4749)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4828..4932
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4933..5001,5002..5793)
                     /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(5151..5170)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5964..6552
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6453..6472
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6706..6723
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6738..6877
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6803..6822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6963..6985
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6979..7170)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7979..8057,1..1004))
                     /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 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
       61 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
      121 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
      181 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
      241 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
      301 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
      361 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
      421 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
      481 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      541 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      601 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      661 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      721 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      781 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      841 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
      901 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
      961 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     1021 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     1081 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1141 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     1201 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     1261 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     1321 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     1381 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     1441 gaaataattt tgtttaactt tggcccaggc ggccaggagg gtctcacatg caggaccggg
     1501 gtaataccgt caccatcgac tttatgtcgg cggatggtat tgttccaggc cgtacgcctg
     1561 ttcgttatca gggcgttgaa gtcggaacag tgcaggatat cagcctcagc gacgatcttc
     1621 gtaagattga agtcaaggtc agcatcaagt ccgatatgaa agatgcgctg cgcgaagaga
     1681 ctcagttctg gctggtgacg ccaaaagcat cgttggcagg tgtctccggg ctggacgccc
     1741 tcgtcggtgg gaactatatc ggcatgatgc cgggtaaagg taaagagcag gatcactttg
     1801 tcgcactcga tacccaaccg aaatatcggc tggacaatgg cgatctgatg atccacctgc
     1861 aagcccccga tctcggttcg ctgaacagcg gttcattggt ctatttccgc aagatcccgg
     1921 tgggaaaagt ctacgactat gccatcaatc ccaacaagca aggcgtggtg attgatgtcc
     1981 tgatcgagcg gcgttttacc gatctggtga aaaaaggtag ccgtttctgg aacgtttccg
     2041 gcgttgatgc caacgtcagt atcagtggcg cgaaggtgaa actggaaagc ctggcggcac
     2101 tggttaacgg tgcgattgcc ttcgattcac cagaagagtc gaaacctgcc gaggcggaag
     2161 atacctttgg tctgtatgaa gatctggccc acagccagcg tggcgtaata ataaaactgg
     2221 aactgccgag tggggccgga ttaaccgccg actcgacgcc gttaatgtat caggggctgg
     2281 aagtcggaca gctgactaaa ctggatttaa atcctggtgg taaagtcaca ggggaaatga
     2341 ccgttgatcc cagcgtcgtt accctgcttc gtgaaaatac ccgcatcgaa ttacgcaacc
     2401 cgaaattatc ccttagcgat gctaatctca gcgccctgct gaccggcaaa accttcgagc
     2461 tggtgcccgg cgatggcgag ccacgcaaag agttcgttgt tgtgccaggc gaaaaagcac
     2521 tgctgcatga acctgatgtt ctgacgctga ccctgaccgc gccggaaagt tacggtattg
     2581 atgcgggtca gccgctcatt cttcacggcg tgcaggtagg ccaggttatc gatcgtaaac
     2641 tcaccagcaa aggcgtcacc tttaccgtcg ccatcgagcc tcagcatcgc gaactggtaa
     2701 aaggcgatag caaatttgtc gtcaacagcc gtgtcgatgt gaaggtgggg ctggatggcg
     2761 ttgagtagct cggtgccagc gcctcagaat ggatcaatgg cgggatacgt attctgccgg
     2821 gcgataaagg tgagatgaaa gccagctatc cactgtatgc caatttggaa aaagcgctgg
     2881 agaacagcct tagcgattta cccaccacaa ccgtgagttt gagtgcagag acgctgccgg
     2941 atgtgcaggc aggatcggta gtgctgtacc gtaaatttga agttggtgaa gtgattaccg
     3001 tgcgtccgcg agctaacgcg tttgatatcg atctgcatat taagccggag tatcgcaacc
     3061 ttctgaccag caatagcgtg ttctgggcag aaggcggggc gaaagttcag ctgaatggta
     3121 gtggtctgac cgtacaggca tccccgctct ccagagcatt aaagggagcc attagcttcg
     3181 acaacctcag cggtgccagc gccagtcagc gtaaaggcga taagcgtatt ctgtatgctt
     3241 ccgaaacagc ggcccgtgcg gttggcgggc agattacgct tcacgctttc gatgccggaa
     3301 aactggcggt cgggatgcca attcgctatc tcggtattga tatcgggcaa atccagacgc
     3361 tggatctgat taccgcacgc aatgaagtac aggcaaaggc ggtactttac ccggaatatg
     3421 tccagacctt tgctcgcggt ggtacgcgct tctcagtggt cacaccgcaa atttcggcag
     3481 ctggcgttga gcatcttgat actatcctcc agccgtatat caacgtcgaa ccaggccggg
     3541 gcaatcctcg ccgcgacttt gaattacaag aggccaccat tactgattcg cgttacctgg
     3601 atggcttaag cattattgtt gaagcgccgg aagccggttc gttaggcatc ggtacgcctg
     3661 tgctgttccg tggtctggaa gtcggtacgg ttacaggaat gacgctgggg acattgtcag
     3721 atcgcgtgat gattgcgatg cgcatcagta aacgctatca acacctggtg cgtaacaatt
     3781 ccgtcttctg gttggcatcg ggttacagtc tggactttgg tctgacgggc ggcgtagtga
     3841 aaaccggcac ctttaaccag tttatccgtg gcggcatcgc cttcgccacg cctccgggta
     3901 cgccactggc accgaaagcc caggaaggca aacacttcct gttgcaggaa agtgaaccga
     3961 aagagtggcg tgaatgggga actgcgcttc ccaaaggcga gacccatcac caccatcacc
     4021 attaaggcca ggccggccga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     4081 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     4141 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     4201 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4261 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4321 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4381 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4441 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4501 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4561 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4621 aaatattaac gcttacaatt taggtggcac ttttcgggga acgacgacct gcagccaagc
     4681 tagcttggct ggacgtaaac tcctcttcag acctaataac ttcgtatagc atacattata
     4741 cgaagttata ttaagggtta ttgaatatga tcggaattgg gctgcaggaa ttcgatagct
     4801 tggctgcagg tcgacctcga ggatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4861 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4921 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4981 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5041 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5101 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5161 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5221 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5281 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5341 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5401 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5461 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5521 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5581 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5641 gagcgtggct ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5701 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5761 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5821 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5881 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5941 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6001 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6061 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6121 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6181 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6241 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6301 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6361 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6421 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6481 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6541 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6601 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6661 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6721 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     6781 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     6841 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     6901 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6961 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     7021 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     7081 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     7141 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     7201 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     7261 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     7321 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     7381 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     7441 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     7501 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     7561 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     7621 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     7681 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     7741 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     7801 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     7861 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     7921 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     7981 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     8041 cgcggggaga ggcggtt
//
