LOCUS       sequence-309627-        8396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for naturally occurring 8xMCE protein (without TM
            region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8396)
  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 8396)
  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: pBEL1755
FEATURES             Location/Qualifiers
     source          1..8396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     126..145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        126..144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    145..169
                     /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             3079..3096
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3162..3180)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3176..3223
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3260..3715
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3347..3366)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3557..3578
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3790..3823)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3902..4006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4007..4075,4076..4867)
                     /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(4225..4244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5038..5626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5527..5546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5780..5797
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5812..5951
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5877..5896)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6037..6059
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6053..6244)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7053..8135)
                     /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     8097..8116
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8136..8213)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
       61 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
      121 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
      181 aattttgttt aactttggcc caggcggcca ggagggtctc acatgggtaa agagatacgt
      241 gttcaatttg acacgggggc aggtttagtg attggaaaaa ccccactgaa gtaccgtggc
      301 attaccattg gcaaggttgt ggactttgag atcgctgatt cgttggataa agtggtcgta
      361 gtcattaagc ttgatcatga ggctgctgga gtggcaaaag ttggtaacaa attttggatt
      421 gtaaagcccg agttaagctt ggatcgtgtc actggtttag aaacgattat ttcagggtca
      481 tacattgagg tccaaccgcc ctcttacgat cttgccgatt tggagtcccg cgacgacgaa
      541 agtttttttg ttgggttgaa agcgcccccg cgctacgaat tctcggggga cgtggttaac
      601 gttaatctga tttctgaatc ggacccgaag ctgtttcgtg ggatgacggt cttccacaat
      661 tctatcgaag ctgggcagat tctgtcggtg acctacgaca acaagaataa catttacttg
      721 gttagtgcga tgatctcgag cgaatatggg cagtatatta atgaaactac taaattttgg
      781 aacatttcag gggtcgatat ctcactggac gcgagtgggt ttaacctgca cacagtgcca
      841 ctgttgggcg cctttcaagg aggggtggcg tttgacgcag aaacgacggg tcgccgtgca
      901 gagccattag atgagtatga tctgtatgat aattacgaag acacgcaact ttcccgtatg
      961 tgtatcacgc ttgacatgcc ggacagctac ggggtcaaaa gtgatcgcac ccccgtaatg
     1021 tttaaaggag tcaaagttgg acttgtcaag gacgttaaat tgaataacga tcgtagtggt
     1081 gccgtcgtga acattatgtt aatgaagaaa taccttgatt tagccgccga gggaagtcgc
     1141 ttcgtgctgc gtcagccatc aatctctgct aaaggtgttg aaaacatcgc cacggcggta
     1201 acaggcgtgt accttgagat tatcgcggga gcgggagaac ctaagtatga attcaccctt
     1261 gctgaggagc ccgtagtgga tgtcccggca gggagtattc caattatcat gactgcagat
     1321 gaaaagggtt cggtcaactc agggtctggg atttacttta aaggcgtgcg cgccggagtc
     1381 gttacgggat accgtttgga agatagccgc gtgatcttcg acgcagtgat tttcaaagaa
     1441 ttccgcgaga ttgccgtgga gggcttatat ttttgggagg ctgatgctct taaggtccgc
     1501 atgtccgggg agggggtttc tattaagaca actccagtgg cccaaatctt agaatcggcc
     1561 gtgaaagcgg ggttctttgg cgctcgtgga aaaaagccgc ttgaacgtga cacggttctt
     1621 gccttatatc ctgacgagga cactgcgaaa gaagcataca ttgcgaaaca aggcatccgt
     1681 aaaatctacc tgatcaccga agatgcgagc tcgctggcta aaggcgcacc cgtcctgtat
     1741 aagggattca aagtggggga acttggaaac cgttttcttg acgcagatac tggcaaggtc
     1801 cgcatttcag cgatgattca accggaatat aaaaaacttg tcaacgacaa aacctacttc
     1861 tggaagttgg gcggctcctc ggtgaagttc gaaggatccg agatcaaagt gtctgcaccc
     1921 gatattaagg aaatcgtctt gggtggggtc gaattcgata atgcagacgc acctcttggg
     1981 agcgttggca gtgttattta tgaagaccac actgatgccc agaaggctgt aaacgcggcc
     2041 ttaagcgccg aaaaagtccg tctttactcc gacaaggaga ctatcacacc cctgggggca
     2101 cctgttttat accgcggcgt gaaagccggg gaggtgtata gtactggcta tgataagaag
     2161 aagaaagccg cttatgcaga tgtaattttg tatcgtgagt tcagcgggac tttaaccgac
     2221 actacgcgct tctggaagtc tgattccttc tccttcgaag cgactgggcg tggagtagat
     2281 atcaaagcag agcccattgc atcttacaca actggagcgt tgaactatga atcattcaag
     2341 aaaccggtag gaacagatat tatttacagc gatcgtggtg cagcggaagc tcccgatctt
     2401 atccgtgtcg aactgcagtt gtctaacgcc tccaatctta aacgcaaggc ccccgtatta
     2461 tgtaacgggg ttcaaatcgg ctatgtcaat actattgcgg gcgacatgac ggctgagttg
     2521 cttatctatg aaaaatatgc ggatttttta cgcgaggggg caaagttctg gattgaagat
     2581 gtgaaagtgt cgttggaggg tttcgagaac gtgaattcga tgatctatgg ggcaaaaatt
     2641 aatatgtttc cgggatctgg tgaggcgctt tttaccttta aaatcgacga taccccagtc
     2701 tcgccttatt atggtcgtaa gggactgcgt ttggtcctgc gctccgactc ccgcagttcc
     2761 ttagagactg ggtcgccggt gtattaccgc caagtacccg ccggcggcgt agaatgggtg
     2821 cgcttggcag acgatggttc ccgcgttgag gtcggtattt tcattgaaga caaataccgc
     2881 catttagtac gttctgtctc tgtattcgag tcggtatcgg gaatcaatgc gcgtttcgga
     2941 ctgttcagtg gctttcgttt gaaaacagga agcgccaaat ctatccttat gggaggggtc
     3001 tcttttacga ctgaaaatat ggacgcgccc gaagctgagg aatcccaagt atttgaactg
     3061 aaaacagacg gcgagaccca tcaccaccat caccattaag gccaggccgg ccgatccggc
     3121 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     3181 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     3241 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3301 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3361 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3421 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3481 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3541 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3601 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3661 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     3721 gcacttttcg gggaacgacg acctgcagcc aagctagctt ggctggacgt aaactcctct
     3781 tcagacctaa taacttcgta tagcatacat tatacgaagt tatattaagg gttattgaat
     3841 atgatcggaa ttgggctgca ggaattcgat agcttggctg caggtcgacc tcgaggatgt
     3901 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3961 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4021 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4081 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4141 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4201 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4261 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4321 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4381 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4441 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4501 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     4561 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4621 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4681 tggctggttt attgctgata aatctggagc cggtgagcgt ggctctcgcg gtatcattgc
     4741 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4801 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4861 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4921 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4981 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5041 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5101 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5161 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5221 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5281 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5341 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5401 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5461 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5521 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5581 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5641 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5701 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5761 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     5821 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     5881 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5941 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6001 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6061 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     6121 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     6181 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     6241 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     6301 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     6361 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     6421 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     6481 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     6541 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     6601 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     6661 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     6721 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     6781 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     6841 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     6901 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     6961 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     7021 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     7081 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     7141 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     7201 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     7261 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     7321 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     7381 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     7441 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     7501 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     7561 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     7621 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     7681 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     7741 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     7801 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     7861 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     7921 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     7981 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     8041 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     8101 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     8161 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     8221 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     8281 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     8341 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagt
//
