LOCUS       sequence-356905-        7703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB Delta-Ring 5 (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7703)
  AUTHORS   Vieni C, Coudray N, Isom GL, Bhabha G, Ekiert DC
  TITLE     Role of Ring6 in the Function of the E. coli MCE Protein LetB.
  JOURNAL   J Mol Biol. 2022 Jan 22;434(7):167463. doi: 
            10.1016/j.jmb.2022.167463.
  PUBMED    35077766
REFERENCE   2  (bases 1 to 7703)
  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"; journalName: "J Mol 
            Biol."; date: "2022-01-22"; volume: "434(7):167463. doi"; pages: " 
            10.1016/j.jmb.2022.167463"
COMMENT     Sequence Label: pBEL2309
FEATURES             Location/Qualifiers
     source          1..7703
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..78
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    93..232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(158..177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     318..340
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(334..525)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1334..2416)
                     /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     2378..2397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2417..2494)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2700..2719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2803..2822
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2803..2821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2822..2846
                     /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             5063..5080
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5146..5164)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5160..5207
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5244..5699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5331..5350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5541..5562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5774..5807)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5886..5990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5991..6059,6060..6851)
                     /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(6209..6228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7022..7610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7511..7530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
       61 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
      121 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
      181 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
      241 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      301 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      361 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
      421 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
      481 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
      541 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
      601 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
      661 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
      721 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
      781 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
      841 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
      901 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
      961 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     1021 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     1081 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     1141 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     1201 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     1261 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     1321 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     1381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     1441 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     1501 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     1561 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     1621 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     1681 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     1741 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     1801 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     1861 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     1921 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     1981 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     2041 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     2101 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     2161 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     2221 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     2281 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     2341 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     2401 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     2461 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     2521 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     2581 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     2641 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     2701 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     2761 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     2821 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttggcccag
     2881 gcggccagga gggtctcaca tgcaggaccg gggtaatacc gtcaccatcg actttatgtc
     2941 ggcggatggt attgttccag gccgtacgcc tgttcgttat cagggcgttg aagtcggaac
     3001 agtgcaggat atcagcctca gcgacgatct tcgtaagatt gaagtcaagg tcagcatcaa
     3061 gtccgatatg aaagatgcgc tgcgcgaaga gactcagttc tggctggtga cgccaaaagc
     3121 atcgttggca ggtgtctccg ggctggacgc cctcgtcggt gggaactata tcggcatgat
     3181 gccgggtaaa ggtaaagagc aggatcactt tgtcgcactc gatacccaac cgaaatatcg
     3241 gctggacaat ggcgatctga tgatccacct gcaagccccc gatctcggtt cgctgaacag
     3301 cggttcattg gtctatttcc gcaagatccc ggtgggaaaa gtctacgact atgccatcaa
     3361 tcccaacaag caaggcgtgg tgattgatgt cctgatcgag cggcgtttta ccgatctggt
     3421 gaaaaaaggt agccgtttct ggaacgtttc cggcgttgat gccaacgtca gtatcagtgg
     3481 cgcgaaggtg aaactggaaa gcctggcggc actggttaac ggtgcgattg ccttcgattc
     3541 accagaagag tcgaaacctg ccgaggcgga agataccttt ggtctgtatg aagatctggc
     3601 ccacagccag cgtggcgtaa taataaaact ggaactgccg agtggggccg gattaaccgc
     3661 cgactcgacg ccgttaatgt atcaggggct ggaagtcgga cagctgacta aactggattt
     3721 aaatcctggt ggtaaagtca caggggaaat gaccgttgat cccagcgtcg ttaccctgct
     3781 tcgtgaaaat acccgcatcg aattacgcaa cccgaaatta tcccttagcg atgctaatct
     3841 cagcgccctg ctgaccggca aaaccttcga gctggtgccc ggcgatggcg agccacgcaa
     3901 agagttcgtt gttgtgccag gcgaaaaagc actgctgcat gaacctgatg ttctgacgct
     3961 gaccctgacc gcgccggaaa gttacggtat tgatgcgggt cagccgctca ttcttcacgg
     4021 cgtgcaggta ggccaggtta tcgatcgtaa actcaccagc aaaggcgtca cctttaccgt
     4081 cgccatcgag cctcagcatc gcgaactggt aaaaggcgat agcaaatttg tcgtcaacag
     4141 ccgtgtcgat gtgaaggtgg ggctggatgg cgttgagttt ctcggtgcca gcgcctcaga
     4201 atggatcaat ggcgggatac gtattctgcc gggcgataaa ggtgagatga aagccagcta
     4261 tccactgtat gccaatttgg aaaaagcgct ggagaacagc cttagcgatt tacccaccac
     4321 aggcgggcag attacgcttc acgctttcga tgccggaaaa ctggcggtcg ggatgccaat
     4381 tcgctatctc ggtattgata tcgggcaaat ccagacgctg gatctgatta ccgcacgcaa
     4441 tgaagtacag gcaaaggcgg tactttaccc ggaatatgtc cagacctttg ctcgcggtgg
     4501 tacgcgcttc tcagtggtca caccgcaaat ttcggcagct ggcgttgagc atcttgatac
     4561 tatcctccag ccgtatatca acgtcgaacc aggccggggc aatcctcgcc gcgactttga
     4621 attacaagag gccaccatta ctgattcgcg ttacctggat ggcttaagca ttattgttga
     4681 agcgccggaa gccggttcgt taggcatcgg tacgcctgtg ctgttccgtg gtctggaagt
     4741 cggtacggtt acaggaatga cgctggggac attgtcagat cgcgtgatga ttgcgatgcg
     4801 catcagtaaa cgctatcaac acctggtgcg taacaattcc gtgttctggt tggcatcggg
     4861 ttacagtctg gactttggtc tgacgggcgg cgtagtgaaa accggcacct ttaaccagtt
     4921 tatccgtggc ggcatcgcct tcgccacgcc tccgggtacg ccactggcac cgaaagccca
     4981 ggaaggcaaa cacttcctgt tgcaggaaag tgaaccgaaa gagtggcgtg aatggggaac
     5041 tgcgcttccc aaaggcgaga cccatcacca ccatcaccat taaggccagg ccggccgatc
     5101 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     5161 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     5221 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5281 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5341 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5401 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5461 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5521 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5581 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5641 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5701 ggtggcactt ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc
     5761 ctcttcagac ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt
     5821 gaatatgatc ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg
     5881 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5941 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6001 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6061 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6121 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6181 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6241 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6301 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6361 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6421 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6481 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     6541 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6601 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6661 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca
     6721 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6781 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6841 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6901 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6961 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7021 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7081 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7141 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     7201 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7261 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7321 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7381 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7441 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7501 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7561 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7621 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7681 cgttatcccc tgattctgtg gat
//
