LOCUS       sequence-309824-        7004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for 4 ring (rings 1347) LetB (without TM region) 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7004)
  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 7004)
  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: pBEL2045
FEATURES             Location/Qualifiers
     source          1..7004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     186..205
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        186..204
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    205..229
                     /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             1747..1764
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1830..1848)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1844..1891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1928..2383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2015..2034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2225..2246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2458..2491)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2570..2674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2675..2743,2744..3535)
                     /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(2893..2912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3706..4294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4195..4214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4448..4465
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4480..4619
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4545..4564)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4705..4727
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4721..4912)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5721..6803)
                     /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     6765..6784
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6804..6881)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
       61 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
      121 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
      181 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
      241 aattttgttt aactttggcc caggcggcca ggagggtctc acatgcagga ccggggtaat
      301 accgtcacca tcgactttat gtcggcggat ggtattgttc caggccgtac gcctgttcgt
      361 tatcagggcg ttgaagtcgg aacagtgcag gatatcagcc tcagcgacga tcttcgtaag
      421 attgaagtca aggtcagcat caagtccgat atgaaagatg cgctgcgcga agagactcag
      481 ttctggctgg tgacgccaaa agcatcgttg gcaggtgtct ccgggctgga cgccctcgtc
      541 ggtgggaact atatcggcat gatgccgggt aaaggtaaag agcaggatca ctttgtcgca
      601 ctcgataccc aaccgaaata tcggctggac aatggcggcg taataataaa actggaactg
      661 ccgagtgggg ccggattaac cgccgactcg acgccgttaa tgtatcaggg gctggaagtc
      721 ggacagctga ctaaactgga tttaaatcct ggtggtaaag tcacagggga aatgaccgtt
      781 gatcccagcg tcgttaccct gcttcgtgaa aatacccgca tcgaattacg caacccgaaa
      841 ttatccctta gcgatgctaa tctcagcgcc ctgctgaccg gcaaaacctt cgagctggtg
      901 cccggcgatg gcgagccacg caaagagttc gttgttgtgc caggcgaaaa agcactgctg
      961 catgaacctg atgttctgac gctgaccctg accgcgccgg aaagttacgg tattgatgcg
     1021 ggtcagccgc tcattcttca cggcgtgcag gtaggccagg ttatcgatcg taaactcacc
     1081 agcaaaggcg tcacctttac cgtcgccatc gagcctcagc atcgcgaact ggtaaaaggc
     1141 gatagcaaat ttgtcgtcaa cagccgtgtc gatgtgaagg tggggctgga tggcgttgag
     1201 tttctcggtg ccagcgcctc agaatggatc aatggcggga tacgtattct gccgggcgat
     1261 aaaggtgaga tgaaagccag ctatccactg tatgccaatt tggaaaaagc gctggagaac
     1321 agccttagcg atttacccac cacaggctta agcattattg ttgaagcgcc ggaagccggt
     1381 tcgttaggca tcggtacgcc tgtgctgttc cgtggtctgg aagtcggtac ggttacagga
     1441 atgacgctgg ggacattgtc agatcgcgtg atgattgcga tgcgcatcag taaacgctat
     1501 caacacctgg tgcgtaacaa ttccgtcttc tggttggcat cgggttacag tctggacttt
     1561 ggtctgacgg gcggcgtagt gaaaaccggc acctttaacc agtttatccg tggcggcatc
     1621 gccttcgcca cgcctccggg tacgccactg gcaccgaaag cccaggaagg caaacacttc
     1681 ctgttgcagg aaagtgaacc gaaagagtgg cgtgaatggg gaactgcgct tcccaaaggc
     1741 gagacccatc accaccatca ccattaaggc caggccggcc gatccggctg ctaacaaagc
     1801 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1861 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1921 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2401 gaacgacgac ctgcagccaa gctagcttgg ctggacgtaa actcctcttc agacctaata
     2461 acttcgtata gcatacatta tacgaagtta tattaagggt tattgaatat gatcggaatt
     2521 gggctgcagg aattcgatag cttggctgca ggtcgacctc gaggatgtgc gcggaacccc
     2581 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2641 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2701 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2761 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2821 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2881 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2941 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3001 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3061 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3121 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3181 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     3241 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3301 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3361 tgctgataaa tctggagccg gtgagcgtgg ctctcgcggt atcattgcag cactggggcc
     3421 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3481 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3541 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3601 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3661 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3721 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3781 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3841 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3901 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3961 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4021 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4081 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4141 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4201 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4261 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4321 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4381 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4441 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4501 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4561 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4621 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4681 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4741 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4801 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4861 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4921 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4981 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5041 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5101 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5161 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     5221 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     5281 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     5341 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     5401 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5461 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5521 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5581 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5641 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5701 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5821 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5881 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5941 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     6001 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     6061 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     6121 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     6181 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     6241 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     6301 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     6361 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6421 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6481 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6541 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6601 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6661 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6721 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6781 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6841 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6901 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6961 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtc
//
