LOCUS       sequence-341986-        8024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB PLL2 deletion (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8024)
  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 8024)
  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: pBEL1994
FEATURES             Location/Qualifiers
     source          1..8024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      64..519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(151..170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     361..382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(594..627)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        706..810
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(811..879,880..1671)
                     /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(1029..1048)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1842..2430
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2331..2350
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2584..2601
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2616..2755
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2681..2700)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2841..2863
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2857..3048)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3857..4939)
                     /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     4901..4920
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4940..5017)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5223..5242
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5326..5345
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5326..5344
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5345..5369
                     /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             7907..7924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7990..8008)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(8004..8024,1..27)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
       61 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      121 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      181 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      241 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      301 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      361 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      421 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      481 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaac
      541 gacgacctgc agccaagcta gcttggctgg acgtaaactc ctcttcagac ctaataactt
      601 cgtatagcat acattatacg aagttatatt aagggttatt gaatatgatc ggaattgggc
      661 tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg aacccctatt
      721 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
      781 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
      841 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
      901 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
      961 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1021 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1081 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1141 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1201 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1261 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1321 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     1381 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     1441 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     1501 gataaatctg gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat
     1561 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1621 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1681 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1741 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1801 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1861 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1921 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1981 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2041 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2101 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2161 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2221 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2281 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2341 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2401 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2461 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2521 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2581 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     2641 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     2701 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     2761 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2821 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2881 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     2941 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3001 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3061 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3121 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3181 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3241 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3301 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3361 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3421 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3481 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3541 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3601 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3661 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     3721 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     3781 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     3841 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3901 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3961 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4021 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4081 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     4141 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4201 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4261 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4321 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4381 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4441 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4501 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4561 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4621 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4681 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4741 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4801 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     4861 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     4921 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4981 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5041 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5101 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5161 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5221 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5281 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     5341 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttggcc
     5401 caggcggcca ggagggtctc acatgcagga ccggggtaat accgtcacca tcgactttat
     5461 gtcggcggat ggtattgttc caggccgtac gcctgttcgt tatcagggcg ttgaagtcgg
     5521 aacagtgcag gacatcagcc tcagcgacga tcttcgtaag attgaagtca aggtcagcat
     5581 caagtccgat atgaaagatg cgctgcgcga agagactcag ttctggctgg tgacgccaaa
     5641 agcatcgttg gcaggtgtct ccgggctgga cgccctcgtc ggtgggaact atatcggcat
     5701 gatgcccggg aaaggtaaag agcaggatca ctttgtcgca ctcgataccc aaccgaaata
     5761 tcggctggac aatggcgatc tgatgatcca cctgcaagcc cccgatctcg gttcgctgaa
     5821 cagcggttca ttggtctatt tccgcaagat cccggtggga aaagtctacg actatgccat
     5881 caatcccaac aagcaaggcg tggtgattga tgtcctgatc gagcggcgtt ttaccgatct
     5941 ggtgaaaaaa ggtagccgtt tctggaacgt ttccggcgtt ggtggggaaa gcttggcggc
     6001 actggttaac ggtgcgattg ccttcgattc accagaagag tcgaaacctg ccgaggcgga
     6061 agataccttt ggtctgtatg aagatctggc ccacagccag cgtggcgtaa taataaaact
     6121 ggaactgccg agtggggccg gattaaccgc cgactcgacg ccgttaatgt atcaggggct
     6181 ggaagtcgga cagctgacta aactggattt aaatcctggt ggtaaagtca caggggaaat
     6241 gaccgttgat cccagcgtcg ttaccctgct tcgtgaaaat acccgcatcg aattacgcaa
     6301 cccgaaatta tcccttagcg atgctaatct cagcgccctg ctgaccggca aaaccttcga
     6361 gctggtgccc ggcgatggcg agccacgcaa agaattcgtt gttgtgccag gcgaaaaagc
     6421 actgctgcat gaacctgatg ttctgacgct gaccctgacc gcgccggaaa gttacggtat
     6481 tgatgcgggt cagccgctca ttcttcacgg cgtgcaggta ggccaggtta tcgatcgtaa
     6541 actcaccagc aaaggcgtca cctttaccgt cgccatcgag cctcagcatc gcgaactggt
     6601 aaaaggcgat agcaaatttg tcgtcaacag ccgtgtcgat gtgaaggtgg ggctggatgg
     6661 cgttgagttt ctcggtgcca gcgcctcaga atggatcaat ggcgggatcc gtattctgcc
     6721 gggcgataaa ggtgagatga aagccagcta tccactgtat gccaatttgg aaaaagcgct
     6781 ggagaacagc cttagcgatt tacccaccac aaccgtgagt ttgagtgcag agacgctgcc
     6841 ggatgtgcag gcaggatcgg tagtgctgta ccgtaaattt gaagttggtg aagtgattac
     6901 cgtgcgtccg cgagctaacg cgtttgacat cgatctgcat attaagccgg agtatcgcaa
     6961 ccttctgacc agcaatagcg tgttctgggc agaaggcggg gcgaaagttc agctgaatgg
     7021 tagtggtctg accgtacagg catccccgct ctccagagct ctaaagggag ccattagctt
     7081 cgacaacctc agcggtgcca gcgccagtca gcgtaaaggc gataagcgta ttctgtatgc
     7141 ttccgaaaca gcggcccgtg cggttggcgg gcagattacg cttcacgctt tcgatgccgg
     7201 aaaactggcg gtcgggatgc caattcgcta tctcggtatt gacatcgggc aaatccagac
     7261 gctggatctg attaccgcac gcaatgaagt acaggcaaag gcggtacttt acccggaata
     7321 tgtccagacc tttgctcgcg gtggtacgcg cttctcagtg gtcacaccgc aaatttcggc
     7381 agctggcgtt gagcatcttg atactatcct ccagccgtat atcaacgtcg aaccaggccg
     7441 gggcaatcct cgccgcgact ttgaattaca agaggccacc attactgatt cgcggtacct
     7501 ggatggctta agcattattg ttgaagcgcc ggaagccggt tcgttaggca tcggtacgcc
     7561 tgtgctgttc cgtggtctgg aagtcggtac ggttacagga atgacgctgg ggacattgtc
     7621 agatcgcgtg atgattgcga tgcgcatcag taaacgctat caacacctgg tgcgtaacaa
     7681 ttccgtcttc tggttggcat cgggttacag tctggacttt ggtctgacgg gcggcgtagt
     7741 gaaaaccggc acctttaacc agtttatccg tggcggcatc gccttcgcca cgcctccggg
     7801 tacgccactg gcaccgaaag cccaggaagg caaacacttc ctgttgcagg aaagtgaacc
     7861 gaaagagtgg cgtgaatggg gaactgcgct tcccaaaggc gagacccatc accaccatca
     7921 ccattaaggc caggccggcc gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     7981 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcc
//
