LOCUS       sequence-309618-        6227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid of rings 2 and 3 of LetB for crystallography.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6227)
  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 6227)
  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: pBEL1584
FEATURES             Location/Qualifiers
     source          1..6227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     199..218
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        199..217
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    218..242
                     /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             983..1000
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1066..1084)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1080..1127
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1164..1619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1251..1270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1461..1482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(1694..1727)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1806..1910
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1911..1979,1980..2771)
                     /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(2129..2148)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2942..3530
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3431..3450
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3684..3701
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3716..3855
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3781..3800)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3941..3963
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3957..4148)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4957..6039)
                     /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     6001..6020
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6040..6117)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
       61 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
      121 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      181 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      241 cccctctaga aataattttg tttaactttg gcccaggcgg ccaggagggt ctcacatggg
      301 cgatctgatg atccacctgc aagcccccga tctcggttcg ctgaacagcg gttcattggt
      361 ctatttccgc aagatcccgg tgggaaaagt ctacgactat gccatcaatc ccaacaagca
      421 aggcgtggtg attgatgtcc tgatcgagcg gcgttttacc gatctggtga aaaaaggtag
      481 ccgtttctgg aacgtttccg gcgttgatgc caacgtcagt atcagtggcg cgaaggtgaa
      541 actggaaagc ctggcggcac tggttaacgg tgcgattgcc ttcgattcac cagaagagtc
      601 gaaacctgcc gaggcggaag atacctttgg tctgtatgaa gatctggccc acagccagcg
      661 tggcgtaata ataaaactgg aactgccgag tggggccgga ttaaccgccg actcgacgcc
      721 gttaatgtat caggggctgg aagtcggaca gctgactaaa ctggatttaa atcctggtgg
      781 taaagtcaca ggggaaatga ccgttgatcc cagcgtcgtt accctgcttc gtgaaaatac
      841 ccgcatcgaa ttacgcaacc cgaaattatc ccttagcgat gctaatctca gcgccctgct
      901 gaccggcaaa accttcgagc tggtgcccgg cgatggcgag ccacgcaaag agttcgttgt
      961 tgtgccaggc gaaggcgaga cccatcacca ccatcaccat taaggccagg ccggccgatc
     1021 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1081 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1141 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1201 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1261 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1321 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1381 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1441 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1501 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1561 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1621 ggtggcactt ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc
     1681 ctcttcagac ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt
     1741 gaatatgatc ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg
     1801 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1861 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1921 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1981 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2041 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2101 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2161 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2221 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2281 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2341 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2401 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     2461 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2521 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2581 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca
     2641 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2701 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2761 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2821 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2881 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2941 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3001 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3061 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3121 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3181 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3241 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3301 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3361 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3421 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3481 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3541 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3601 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3661 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3721 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3781 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3841 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3901 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3961 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4021 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4081 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4141 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4201 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4261 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4321 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4381 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4441 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4501 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4561 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4621 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4681 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4741 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4801 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4861 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4921 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4981 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5041 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5101 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5161 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5221 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5281 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5341 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5401 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5461 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5521 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5581 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5641 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5701 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5761 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5821 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5881 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5941 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6001 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6061 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6121 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6181 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatg
//
