LOCUS       sequence-309745-        8027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB PLL5 deletion (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8027)
  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 8027)
  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: pBEL1997
FEATURES             Location/Qualifiers
     source          1..8027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     177..196
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        177..195
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    196..220
                     /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             2761..2778
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2844..2862)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2858..2905
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2942..3397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3029..3048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3239..3260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3472..3505)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3584..3688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3689..3757,3758..4549)
                     /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(3907..3926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4720..5308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5209..5228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5462..5479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5494..5633
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5559..5578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5719..5741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5735..5926)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6735..7817)
                     /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     7779..7798
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7818..7895)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
       61 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      241 taactttggc ccaggcggcc aggagggtct cacatgcagg accggggtaa taccgtcacc
      301 atcgacttta tgtcggcgga tggtattgtt ccaggccgta cgcctgttcg ttatcagggc
      361 gttgaagtcg gaacagtgca ggacatcagc ctcagcgacg atcttcgtaa gattgaagtc
      421 aaggtcagca tcaagtccga tatgaaagat gcgctgcgcg aagagactca gttctggctg
      481 gtgacgccaa aagcatcgtt ggcaggtgtc tccgggctgg acgccctcgt cggtgggaac
      541 tatatcggca tgatgcccgg gaaaggtaaa gagcaggatc actttgtcgc actcgatacc
      601 caaccgaaat atcggctgga caatggcgat ctgatgatcc acctgcaagc ccccgatctc
      661 ggttcgctga acagcggttc attggtctat ttccgcaaga tcccggtggg aaaagtctac
      721 gactatgcca tcaatcccaa caagcaaggc gtggtgattg atgtcctgat cgagcggcgt
      781 tttaccgatc tggtgaaaaa aggtagccgt ttctggaacg tttccggcgt tgatgccaac
      841 gtcagtatca gtggcgcgaa ggtgaaactg gaaagcttgg cggcactggt taacggtgcg
      901 attgccttcg attcaccaga agagtcgaaa cctgccgagg cggaagatac ctttggtctg
      961 tatgaagatc tggcccacag ccagcgtggc gtaataataa aactggaact gccgagtggg
     1021 gccggattaa ccgccgactc gacgccgtta atgtatcagg ggctggaagt cggacagctg
     1081 actaaactgg atttaaatcc tggtggtaaa gtcacagggg aaatgaccgt tgatcccagc
     1141 gtcgttaccc tgcttcgtga aaatacccgc atcgaattac gcaacccgaa attatccctt
     1201 agcgatgcta atctcagcgc cctgctgacc ggcaaaacct tcgagctggt gcccggcgat
     1261 ggcgagccac gcaaagaatt cgttgttgtg ccaggcgaaa aagcactgct gcatgaacct
     1321 gatgttctga cgctgaccct gaccgcgccg gaaagttacg gtattgatgc gggtcagccg
     1381 ctcattcttc acggcgtgca ggtaggccag gttatcgatc gtaaactcac cagcaaaggc
     1441 gtcaccttta ccgtcgccat cgagcctcag catcgcgaac tggtaaaagg cgatagcaaa
     1501 tttgtcgtca acagccgtgt cgatgtgaag gtggggctgg atggcgttga gtttctcggt
     1561 gccagcgcct cagaatggat caatggcggg atccgtattc tgccgggcga taaaggtgag
     1621 atgaaagcca gctatccact gtatgccaat ttggaaaaag cgctggagaa cagccttagc
     1681 gatttaccca ccacaaccgt gagtttgagt gcagagacgc tgccggatgt gcaggcagga
     1741 tcggtagtgc tgtaccgtaa atttgaagtt ggtgaagtga ttaccgtgcg tccgcgagct
     1801 aacgcgtttg acatcgatct gcatattaag ccggagtatc gcaaccttct gaccagcaat
     1861 agcgtgttct gggcagaagg cggggcggga ggggcatccc cgctctccag agctctaaag
     1921 ggagccatta gcttcgacaa cctcagcggt gccagcgcca gtcagcgtaa aggcgataag
     1981 cgtattctgt atgcttccga aacagcggcc cgtgcggttg gcgggcagat tacgcttcac
     2041 gctttcgatg ccggaaaact ggcggtcggg atgccaattc gctatctcgg tattgacatc
     2101 gggcaaatcc agacgctgga tctgattacc gcacgcaatg aagtacaggc aaaggcggta
     2161 ctttacccgg aatatgtcca gacctttgct cgcggtggta cgcgcttctc agtggtcaca
     2221 ccgcaaattt cggcagctgg cgttgagcat cttgatacta tcctccagcc gtatatcaac
     2281 gtcgaaccag gccggggcaa tcctcgccgc gactttgaat tacaagaggc caccattact
     2341 gattcgcggt acctggatgg cttaagcatt attgttgaag cgccggaagc cggttcgtta
     2401 ggcatcggta cgcctgtgct gttccgtggt ctggaagtcg gtacggttac aggaatgacg
     2461 ctggggacat tgtcagatcg cgtgatgatt gcgatgcgca tcagtaaacg ctatcaacac
     2521 ctggtgcgta acaattccgt cttctggttg gcatcgggtt acagtctgga ctttggtctg
     2581 acgggcggcg tagtgaaaac cggcaccttt aaccagttta tccgtggcgg catcgccttc
     2641 gccacgcctc cgggtacgcc actggcaccg aaagcccagg aaggcaaaca cttcctgttg
     2701 caggaaagtg aaccgaaaga gtggcgtgaa tggggaactg cgcttcccaa aggcgagacc
     2761 catcaccacc atcaccatta aggccaggcc ggccgatccg gctgctaaca aagcccgaaa
     2821 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2881 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     2941 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3001 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3061 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3121 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3181 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3241 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3301 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3361 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaacga
     3421 cgacctgcag ccaagctagc ttggctggac gtaaactcct cttcagacct aataacttcg
     3481 tatagcatac attatacgaa gttatattaa gggttattga atatgatcgg aattgggctg
     3541 caggaattcg atagcttggc tgcaggtcga cctcgaggat gtgcgcggaa cccctatttg
     3601 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3661 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3721 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3781 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3841 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3901 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3961 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4021 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4081 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4141 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4201 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     4261 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4321 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4381 taaatctgga gccggtgagc gtggctctcg cggtatcatt gcagcactgg ggccagatgg
     4441 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4501 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4561 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4621 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4681 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4741 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4801 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4861 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4921 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4981 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5041 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5101 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5161 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5221 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5281 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5341 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5401 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5461 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5521 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5581 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5641 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5701 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5761 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5821 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5881 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5941 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     6001 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     6061 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     6121 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     6181 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     6241 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     6301 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     6361 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     6421 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     6481 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     6541 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     6601 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     6661 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     6721 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6781 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6841 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6901 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6961 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     7021 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     7081 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     7141 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     7201 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     7261 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     7321 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     7381 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     7441 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     7501 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     7561 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     7621 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     7681 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7741 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7801 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7861 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     7921 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     7981 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggcca
//
