LOCUS       sequence-309710-        6635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 3 ring (rings 167) LetB (without 
            TM region) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  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 6635)
  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: pBEL2001
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..57
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     141..160
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        141..159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    160..184
                     /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             1333..1350
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1416..1434)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1430..1477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1514..1969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1601..1620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1811..1832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2044..2077)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2156..2260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2261..2329,2330..3121)
                     /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(2479..2498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3292..3880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3781..3800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4034..4051
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4066..4205
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4131..4150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4291..4313
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4307..4498)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5307..6389)
                     /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     6351..6370
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6390..6467)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
       61 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      121 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      181 ttcccctcta gaaataattt tgtttaactt tggcccaggc ggccaggagg gtctcacatg
      241 caggaccggg gtaataccgt caccatcgac tttatgtcgg cggatggtat tgttccaggc
      301 cgtacgcctg ttcgttatca gggcgttgaa gtcggaacag tgcaggatat cagcctcagc
      361 gacgatcttc gtaagattga agtcaaggtc agcatcaagt ccgatatgaa agatgcgctg
      421 cgcgaagaga ctcagttctg gctggtgacg ccaaaagcat cgttggcagg tgtctccggg
      481 ctggacgccc tcgtcggtgg gaactatatc ggcatgatgc cgggtaaagg taaagagcag
      541 gatcactttg tcgcactcga tacccaaccg aaatatcggc tggacaatgg cgatgggcag
      601 attacgcttc acgctttcga tgccggaaaa ctggcggtcg ggatgccaat tcgctatctc
      661 ggtattgata tcgggcaaat ccagacgctg gatctgatta ccgcacgcaa tgaagtacag
      721 gcaaaggcgg tactttaccc ggaatatgtc cagacctttg ctcgcggtgg tacgcgcttc
      781 tcagtggtca caccgcaaat ttcggcagct ggcgttgagc atcttgatac tatcctccag
      841 ccgtatatca acgtcgaacc aggccggggc aatcctcgcc gcgactttga attacaagag
      901 gccaccatta ctgattcgcg ttacctggat ggcttaagca ttattgttga agcgccggaa
      961 gccggttcgt taggcatcgg tacgcctgtg ctgttccgtg gtctggaagt cggtacggtt
     1021 acaggaatga cgctggggac attgtcagat cgcgtgatga ttgcgatgcg catcagtaaa
     1081 cgctatcaac acctggtgcg taacaattcc gtcttctggt tggcatcggg ttacagtctg
     1141 gactttggtc tgacgggcgg cgtagtgaaa accggcacct ttaaccagtt tatccgtggc
     1201 ggcatcgcct tcgccacgcc tccgggtacg ccactggcac cgaaagccca ggaaggcaaa
     1261 cacttcctgt tgcaggaaag tgaaccgaaa gagtggcgtg aatggggaac tgcgcttccc
     1321 aaaggcgaga cccatcacca ccatcaccat taaggccagg ccggccgatc cggctgctaa
     1381 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1441 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1501 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1561 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1621 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1681 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1741 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1801 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1861 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1921 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1981 ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc ctcttcagac
     2041 ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt gaatatgatc
     2101 ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg
     2161 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2221 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2281 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2341 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2401 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2461 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     2521 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2581 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2641 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2701 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2761 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2821 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2881 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2941 gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca ttgcagcact
     3001 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3061 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3121 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3421 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3901 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3961 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4021 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4081 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     4141 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4201 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4261 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4321 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4381 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4441 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4501 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4561 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4621 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4681 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4741 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4801 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4861 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4921 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4981 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     5041 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     5101 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     5161 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     5221 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5281 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5341 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     5401 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     5461 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     5521 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5581 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5641 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5701 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5761 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5821 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5881 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5941 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     6001 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     6061 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     6121 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6181 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     6241 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     6301 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     6361 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     6421 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     6481 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6541 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6601 ggggcctgcc accataccca cgccgaaaca agcgc
//
