LOCUS       sequence-309823-        6992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for 4 ring (rings 1237) LetB (without TM region) 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6992)
  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 6992)
  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: pBEL2044
FEATURES             Location/Qualifiers
     source          1..6992
                     /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             2940..2957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3023..3041)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3037..3084
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3121..3576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3208..3227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3418..3439
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3651..3684)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3763..3867
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3868..3936,3937..4728)
                     /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(4086..4105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4899..5487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5388..5407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5641..5658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5673..5812
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5738..5757)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5898..5920
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5914..6105)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6914..6992,1..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 acctgatggc ttaagcatta ttgttgaagc gccggaagcc ggttcgttag
     2581 gcatcggtac gcctgtgctg ttccgtggtc tggaagtcgg tacggttaca ggaatgacgc
     2641 tggggacatt gtcagatcgc gtgatgattg cgatgcgcat cagtaaacgc tatcaacacc
     2701 tggtgcgtaa caattccgtc ttctggttgg catcgggtta cagtctggac tttggtctga
     2761 cgggcggcgt agtgaaaacc ggcaccttta accagtttat ccgtggcggc atcgccttcg
     2821 ccacgcctcc gggtacgcca ctggcaccga aagcccagga aggcaaacac ttcctgttgc
     2881 aggaaagtga accgaaagag tggcgtgaat ggggaactgc gcttcccaaa ggcgagaccc
     2941 atcaccacca tcaccattaa ggccaggccg gccgatccgg ctgctaacaa agcccgaaag
     3001 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3061 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     3121 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3181 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3241 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3301 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3361 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3421 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3481 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3541 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaacgac
     3601 gacctgcagc caagctagct tggctggacg taaactcctc ttcagaccta ataacttcgt
     3661 atagcataca ttatacgaag ttatattaag ggttattgaa tatgatcgga attgggctgc
     3721 aggaattcga tagcttggct gcaggtcgac ctcgaggatg tgcgcggaac ccctatttgt
     3781 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3841 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3901 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3961 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4021 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4081 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4141 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4201 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4261 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4321 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4381 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4441 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4501 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4561 aaatctggag ccggtgagcg tggctctcgc ggtatcattg cagcactggg gccagatggt
     4621 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4681 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4741 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4801 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4861 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4921 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4981 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5041 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5101 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5161 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5221 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5281 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5341 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5401 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5461 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5521 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5581 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5641 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5701 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5761 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5821 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5881 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5941 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6001 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6061 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     6121 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6181 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     6241 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6301 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6361 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6421 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6481 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6541 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     6601 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     6661 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     6721 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     6781 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     6841 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     6901 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6961 atgaatcggc caacgcgcgg ggagaggcgg tt
//
