LOCUS       sequence-309739-        8015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB PLL3 deletion (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8015)
  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 8015)
  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: pBEL1995
FEATURES             Location/Qualifiers
     source          1..8015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             3967..3984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4050..4068)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4064..4111
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4148..4603
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4235..4254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4445..4466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4678..4711)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4790..4894
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4895..4963,4964..5755)
                     /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(5113..5132)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5926..6514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6415..6434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6668..6685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6700..6839
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6765..6784)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6925..6947
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6941..7132)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7941..8015,1..1008))
                     /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 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttggccc aggcggccag gagggtctca catgcaggac
     1501 cggggtaata ccgtcaccat cgactttatg tcggcggatg gtattgttcc aggccgtacg
     1561 cctgttcgtt atcagggcgt tgaagtcgga acagtgcagg acatcagcct cagcgacgat
     1621 cttcgtaaga ttgaagtcaa ggtcagcatc aagtccgata tgaaagatgc gctgcgcgaa
     1681 gagactcagt tctggctggt gacgccaaaa gcatcgttgg caggtgtctc cgggctggac
     1741 gccctcgtcg gtgggaacta tatcggcatg atgcccggga aaggtaaaga gcaggatcac
     1801 tttgtcgcac tcgataccca accgaaatat cggctggaca atggcgatct gatgatccac
     1861 ctgcaagccc ccgatctcgg ttcgctgaac agcggttcat tggtctattt ccgcaagatc
     1921 ccggtgggaa aagtctacga ctatgccatc aatcccaaca agcaaggcgt ggtgattgat
     1981 gtcctgatcg agcggcgttt taccgatctg gtgaaaaaag gtagccgttt ctggaacgtt
     2041 tccggcgttg atgccaacgt cagtatcagt ggcgcgaagg tgaaactgga aagcttggcg
     2101 gcactggtta acggtgcgat tgccttcgat tcaccagaag agtcgaaacc tgccgaggcg
     2161 gaagatacct ttggtctgta tgaagatctg gcccacagcc agcgtggcgt aataataaaa
     2221 ctggaactgc cgagtggggc cggattaacc gccgactcga cgccgttaat gtatcagggg
     2281 ctggaagtcg gacagctgac taaactggat ttaaatcctg gtggtaaagt cacaggggaa
     2341 atgaccgttg atcccagcgt cgttaccctg cttcgtgaaa atacccgcat cgaattacgc
     2401 aacgggggca aaaccttcga gctggtgccc ggcgatggcg agccacgcaa agaattcgtt
     2461 gttgtgccag gcgaaaaagc actgctgcat gaacctgatg ttctgacgct gaccctgacc
     2521 gcgccggaaa gttacggtat tgatgcgggt cagccgctca ttcttcacgg cgtgcaggta
     2581 ggccaggtta tcgatcgtaa actcaccagc aaaggcgtca cctttaccgt cgccatcgag
     2641 cctcagcatc gcgaactggt aaaaggcgat agcaaatttg tcgtcaacag ccgtgtcgat
     2701 gtgaaggtgg ggctggatgg cgttgagttt ctcggtgcca gcgcctcaga atggatcaat
     2761 ggcgggatcc gtattctgcc gggcgataaa ggtgagatga aagccagcta tccactgtat
     2821 gccaatttgg aaaaagcgct ggagaacagc cttagcgatt tacccaccac aaccgtgagt
     2881 ttgagtgcag agacgctgcc ggatgtgcag gcaggatcgg tagtgctgta ccgtaaattt
     2941 gaagttggtg aagtgattac cgtgcgtccg cgagctaacg cgtttgacat cgatctgcat
     3001 attaagccgg agtatcgcaa ccttctgacc agcaatagcg tgttctgggc agaaggcggg
     3061 gcgaaagttc agctgaatgg tagtggtctg accgtacagg catccccgct ctccagagct
     3121 ctaaagggag ccattagctt cgacaacctc agcggtgcca gcgccagtca gcgtaaaggc
     3181 gataagcgta ttctgtatgc ttccgaaaca gcggcccgtg cggttggcgg gcagattacg
     3241 cttcacgctt tcgatgccgg aaaactggcg gtcgggatgc caattcgcta tctcggtatt
     3301 gacatcgggc aaatccagac gctggatctg attaccgcac gcaatgaagt acaggcaaag
     3361 gcggtacttt acccggaata tgtccagacc tttgctcgcg gtggtacgcg cttctcagtg
     3421 gtcacaccgc aaatttcggc agctggcgtt gagcatcttg atactatcct ccagccgtat
     3481 atcaacgtcg aaccaggccg gggcaatcct cgccgcgact ttgaattaca agaggccacc
     3541 attactgatt cgcggtacct ggatggctta agcattattg ttgaagcgcc ggaagccggt
     3601 tcgttaggca tcggtacgcc tgtgctgttc cgtggtctgg aagtcggtac ggttacagga
     3661 atgacgctgg ggacattgtc agatcgcgtg atgattgcga tgcgcatcag taaacgctat
     3721 caacacctgg tgcgtaacaa ttccgtcttc tggttggcat cgggttacag tctggacttt
     3781 ggtctgacgg gcggcgtagt gaaaaccggc acctttaacc agtttatccg tggcggcatc
     3841 gccttcgcca cgcctccggg tacgccactg gcaccgaaag cccaggaagg caaacacttc
     3901 ctgttgcagg aaagtgaacc gaaagagtgg cgtgaatggg gaactgcgct tcccaaaggc
     3961 gagacccatc accaccatca ccattaaggc caggccggcc gatccggctg ctaacaaagc
     4021 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     4081 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     4141 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4201 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4261 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4321 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4381 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4441 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4501 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4561 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     4621 gaacgacgac ctgcagccaa gctagcttgg ctggacgtaa actcctcttc agacctaata
     4681 acttcgtata gcatacatta tacgaagtta tattaagggt tattgaatat gatcggaatt
     4741 gggctgcagg aattcgatag cttggctgca ggtcgacctc gaggatgtgc gcggaacccc
     4801 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4861 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4921 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4981 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5041 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5101 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5161 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5221 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5281 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5341 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5401 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     5461 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5521 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5581 tgctgataaa tctggagccg gtgagcgtgg ctctcgcggt atcattgcag cactggggcc
     5641 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5701 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5761 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5821 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5881 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5941 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6001 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6061 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6121 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6181 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6241 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6301 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6361 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6421 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6481 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6541 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6601 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6661 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     6721 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     6781 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     6841 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6901 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6961 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     7021 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     7081 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     7141 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     7201 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     7261 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     7321 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     7381 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     7441 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     7501 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     7561 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     7621 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     7681 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     7741 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     7801 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     7861 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     7921 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7981 tgcattaatg aatcggccaa cgcgcgggga gaggc
//
