LOCUS       sequence-309616-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for WT LetB (without TM region) for cryo and 
            negative stain EM.
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: pBEL1324
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     976..995
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1015..1092)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1298..1317
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1401..1420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1401..1419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1420..1444
                     /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             4015..4032
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4098..4116)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4112..4159
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4196..4651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4283..4302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4493..4514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4726..4759)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4838..4942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4943..5011,5012..5803)
                     /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(5161..5180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5974..6562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6463..6482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6716..6733
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6748..6887
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6813..6832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6973..6995
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6989..7180)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7989..8057,1..1014))
                     /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 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
       61 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
      121 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
      181 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
      241 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
      301 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
      361 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
      421 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
      481 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
      541 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
      601 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
      661 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
      721 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
      781 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
      841 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
      901 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
      961 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     1021 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     1081 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     1141 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     1201 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     1261 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     1321 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     1381 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1441 ttcccctcta gaaataattt tgtttaactt tggcccaggc ggccaggagg gtctcacatg
     1501 caggaccggg gtaataccgt caccatcgac tttatgtcgg cggatggtat tgttccaggc
     1561 cgtacgcctg ttcgttatca gggcgttgaa gtcggaacag tgcaggatat cagcctcagc
     1621 gacgatcttc gtaagattga agtcaaggtc agcatcaagt ccgatatgaa agatgcgctg
     1681 cgcgaagaga ctcagttctg gctggtgacg ccaaaagcat cgttggcagg tgtctccggg
     1741 ctggacgccc tcgtcggtgg gaactatatc ggcatgatgc cgggtaaagg taaagagcag
     1801 gatcactttg tcgcactcga tacccaaccg aaatatcggc tggacaatgg cgatctgatg
     1861 atccacctgc aagcccccga tctcggttcg ctgaacagcg gttcattggt ctatttccgc
     1921 aagatcccgg tgggaaaagt ctacgactat gccatcaatc ccaacaagca aggcgtggtg
     1981 attgatgtcc tgatcgagcg gcgttttacc gatctggtga aaaaaggtag ccgtttctgg
     2041 aacgtttccg gcgttgatgc caacgtcagt atcagtggcg cgaaggtgaa actggaaagc
     2101 ctggcggcac tggttaacgg tgcgattgcc ttcgattcac cagaagagtc gaaacctgcc
     2161 gaggcggaag atacctttgg tctgtatgaa gatctggccc acagccagcg tggcgtaata
     2221 ataaaactgg aactgccgag tggggccgga ttaaccgccg actcgacgcc gttaatgtat
     2281 caggggctgg aagtcggaca gctgactaaa ctggatttaa atcctggtgg taaagtcaca
     2341 ggggaaatga ccgttgatcc cagcgtcgtt accctgcttc gtgaaaatac ccgcatcgaa
     2401 ttacgcaacc cgaaattatc ccttagcgat gctaatctca gcgccctgct gaccggcaaa
     2461 accttcgagc tggtgcccgg cgatggcgag ccacgcaaag agttcgttgt tgtgccaggc
     2521 gaaaaagcac tgctgcatga acctgatgtt ctgacgctga ccctgaccgc gccggaaagt
     2581 tacggtattg atgcgggtca gccgctcatt cttcacggcg tgcaggtagg ccaggttatc
     2641 gatcgtaaac tcaccagcaa aggcgtcacc tttaccgtcg ccatcgagcc tcagcatcgc
     2701 gaactggtaa aaggcgatag caaatttgtc gtcaacagcc gtgtcgatgt gaaggtgggg
     2761 ctggatggcg ttgagtttct cggtgccagc gcctcagaat ggatcaatgg cgggatacgt
     2821 attctgccgg gcgataaagg tgagatgaaa gccagctatc cactgtatgc caatttggaa
     2881 aaagcgctgg agaacagcct tagcgattta cccaccacaa ccgtgagttt gagtgcagag
     2941 acgctgccgg atgtgcaggc aggatcggta gtgctgtacc gtaaatttga agttggtgaa
     3001 gtgattaccg tgcgtccgcg agctaacgcg tttgatatcg atctgcatat taagccggag
     3061 tatcgcaacc ttctgaccag caatagcgtg ttctgggcag aaggcggggc gaaagttcag
     3121 ctgaatggta gtggtctgac cgtacaggca tccccgctct ccagagcatt aaagggagcc
     3181 attagcttcg acaacctcag cggtgccagc gccagtcagc gtaaaggcga taagcgtatt
     3241 ctgtatgctt ccgaaacagc ggcccgtgcg gttggcgggc agattacgct tcacgctttc
     3301 gatgccggaa aactggcggt cgggatgcca attcgctatc tcggtattga tatcgggcaa
     3361 atccagacgc tggatctgat taccgcacgc aatgaagtac aggcaaaggc ggtactttac
     3421 ccggaatatg tccagacctt tgctcgcggt ggtacgcgct tctcagtggt cacaccgcaa
     3481 atttcggcag ctggcgttga gcatcttgat actatcctcc agccgtatat caacgtcgaa
     3541 ccaggccggg gcaatcctcg ccgcgacttt gaattacaag aggccaccat tactgattcg
     3601 cgttacctgg atggcttaag cattattgtt gaagcgccgg aagccggttc gttaggcatc
     3661 ggtacgcctg tgctgttccg tggtctggaa gtcggtacgg ttacaggaat gacgctgggg
     3721 acattgtcag atcgcgtgat gattgcgatg cgcatcagta aacgctatca acacctggtg
     3781 cgtaacaatt ccgtcttctg gttggcatcg ggttacagtc tggactttgg tctgacgggc
     3841 ggcgtagtga aaaccggcac ctttaaccag tttatccgtg gcggcatcgc cttcgccacg
     3901 cctccgggta cgccactggc accgaaagcc caggaaggca aacacttcct gttgcaggaa
     3961 agtgaaccga aagagtggcg tgaatgggga actgcgcttc ccaaaggcga gacccatcac
     4021 caccatcacc attaaggcca ggccggccga tccggctgct aacaaagccc gaaaggaagc
     4081 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     4141 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     4201 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4261 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4321 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4381 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4441 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4501 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4561 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4621 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga acgacgacct
     4681 gcagccaagc tagcttggct ggacgtaaac tcctcttcag acctaataac ttcgtatagc
     4741 atacattata cgaagttata ttaagggtta ttgaatatga tcggaattgg gctgcaggaa
     4801 ttcgatagct tggctgcagg tcgacctcga ggatgtgcgc ggaaccccta tttgtttatt
     4861 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4921 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4981 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5041 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5101 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5161 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5221 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5281 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5341 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5401 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5461 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     5521 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5581 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5641 tggagccggt gagcgtggct ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5701 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5761 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5821 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5881 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5941 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6001 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6061 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6121 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6181 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6241 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6301 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6361 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6421 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6481 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6541 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6601 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6661 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6721 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     6781 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6841 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     6901 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     6961 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     7021 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     7081 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     7141 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     7201 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     7261 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     7321 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     7381 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     7441 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     7501 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     7561 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     7621 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     7681 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     7741 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     7801 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     7861 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     7921 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     7981 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     8041 tcggccaacg cgcgggg
//
