LOCUS       sequence-309663-        6296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 2 ring (rings17) LetB (without TM 
            region) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6296)
  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 6296)
  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: pBEL2000
FEATURES             Location/Qualifiers
     source          1..6296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    148..172
                     /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             982..999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1065..1083)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1079..1126
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1163..1618
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1250..1269)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1460..1481
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(1693..1726)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1805..1909
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1910..1978,1979..2770)
                     /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(2128..2147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2941..3529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3430..3449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3683..3700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3715..3854
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3780..3799)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3940..3962
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3956..4147)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4956..6038)
                     /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     6000..6019
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6039..6116)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
       61 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      121 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      181 aataattttg tttaactttg gcccaggcgg ccaggagggt ctcacatgca ggaccggggt
      241 aataccgtca ccatcgactt tatgtcggcg gatggtattg ttccaggccg tacgcctgtt
      301 cgttatcagg gcgttgaagt cggaacagtg caggatatca gcctcagcga cgatcttcgt
      361 aagattgaag tcaaggtcag catcaagtcc gatatgaaag atgcgctgcg cgaagagact
      421 cagttctggc tggtgacgcc aaaagcatcg ttggcaggtg tctccgggct ggacgccctc
      481 gtcggtggga actatatcgg catgatgccg ggtaaaggta aagagcagga tcactttgtc
      541 gcactcgata cccaaccgaa atatcggctg gacaatggcg atttaagcat tattgttgaa
      601 gcgccggaag ccggttcgtt aggcatcggt acgcctgtgc tgttccgtgg tctggaagtc
      661 ggtacggtta caggaatgac gctggggaca ttgtcagatc gcgtgatgat tgcgatgcgc
      721 atcagtaaac gctatcaaca cctggtgcgt aacaattccg tcttctggtt ggcatcgggt
      781 tacagtctgg actttggtct gacgggcggc gtagtgaaaa ccggcacctt taaccagttt
      841 atccgtggcg gcatcgcctt cgccacgcct ccgggtacgc cactggcacc gaaagcccag
      901 gaaggcaaac acttcctgtt gcaggaaagt gaaccgaaag agtggcgtga atggggaact
      961 gcgcttccca aaggcgagac ccatcaccac catcaccatt aaggccaggc cggccgatcc
     1021 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1081 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1141 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1201 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1261 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1321 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1381 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1441 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     1501 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1561 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     1621 gtggcacttt tcggggaacg acgacctgca gccaagctag cttggctgga cgtaaactcc
     1681 tcttcagacc taataacttc gtatagcata cattatacga agttatatta agggttattg
     1741 aatatgatcg gaattgggct gcaggaattc gatagcttgg ctgcaggtcg acctcgagga
     1801 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1861 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1921 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1981 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2041 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2101 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2161 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2221 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2281 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2341 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2401 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2461 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2521 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2581 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat
     2641 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2701 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2761 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2821 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2881 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2941 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3001 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3061 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3121 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3181 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3241 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3301 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3361 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3421 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3481 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3541 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3601 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3661 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3721 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3781 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3841 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3901 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3961 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4021 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4081 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4141 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4201 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4261 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4321 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4381 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4441 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4501 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4561 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4621 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4681 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4741 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4801 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4861 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4921 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4981 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5041 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5101 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5161 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5221 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5281 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5341 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5401 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5461 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5521 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5581 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5641 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5701 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5761 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5821 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5881 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5941 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6001 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6061 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6121 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6181 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6241 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccga
//
