LOCUS       sequence-356906-        7685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB Delta-Ring 4 (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7685)
  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 7685)
  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: pBEL2308
FEATURES             Location/Qualifiers
     source          1..7685
                     /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             5045..5062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5128..5146)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5142..5189
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5226..5681
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5313..5332)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5523..5544
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5756..5789)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5868..5972
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5973..6041,6042..6833)
                     /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(6191..6210)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7004..7592
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7493..7512
                     /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 gaacctgata ccgtgagttt
     3961 gagtgcagag acgctgccgg atgtgcaggc aggatcggta gtgctgtacc gtaaatttga
     4021 agttggtgaa gtgattaccg tgcgtccgcg agctaacgcg tttgatatcg atctgcatat
     4081 taagccggag tatcgcaacc ttctgaccag caatagcgtg ttctgggcag aaggcggggc
     4141 gaaagttcag ctgaatggta gtggtctgac cgtacaggca tccccgctct ccagagcatt
     4201 aaagggagcc attagcttcg acaacctcag cggtgccagc gccagtcagc gtaaaggcga
     4261 taagcgtatt ctgtatgctt ccgaaacagc ggcccgtgcg gttggcgggc agattacgct
     4321 tcacgctttc gatgccggaa aactggcggt cgggatgcca attcgctatc tcggtattga
     4381 tatcgggcaa atccagacgc tggatctgat taccgcacgc aatgaagtac aggcaaaggc
     4441 ggtactttac ccggaatatg tccagacctt tgctcgcggt ggtacgcgct tctcagtggt
     4501 cacaccgcaa atttcggcag ctggcgttga gcatcttgat actatcctcc agccgtatat
     4561 caacgtcgaa ccaggccggg gcaatcctcg ccgcgacttt gaattacaag aggccaccat
     4621 tactgattcg cgttacctgg atggcttaag cattattgtt gaagcgccgg aagccggttc
     4681 gttaggcatc ggtacgcctg tgctgttccg tggtctggaa gtcggtacgg ttacaggaat
     4741 gacgctgggg acattgtcag atcgcgtgat gattgcgatg cgcatcagta aacgctatca
     4801 acacctggtg cgtaacaatt ccgtgttctg gttggcatcg ggttacagtc tggactttgg
     4861 tctgacgggc ggcgtagtga aaaccggcac ctttaaccag tttatccgtg gcggcatcgc
     4921 cttcgccacg cctccgggta cgccactggc accgaaagcc caggaaggca aacacttcct
     4981 gttgcaggaa agtgaaccga aagagtggcg tgaatgggga actgcgcttc ccaaaggcga
     5041 gacccatcac caccatcacc attaaggcca ggccggccga tccggctgct aacaaagccc
     5101 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     5161 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     5221 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5281 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5341 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5401 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5461 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5521 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5581 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5641 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5701 acgacgacct gcagccaagc tagcttggct ggacgtaaac tcctcttcag acctaataac
     5761 ttcgtatagc atacattata cgaagttata ttaagggtta ttgaatatga tcggaattgg
     5821 gctgcaggaa ttcgatagct tggctgcagg tcgacctcga ggatgtgcgc ggaaccccta
     5881 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5941 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6001 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6061 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6121 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6181 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6241 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6301 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6361 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6421 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6481 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     6541 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6601 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6661 ctgataaatc tggagccggt gagcgtggct ctcgcggtat cattgcagca ctggggccag
     6721 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6781 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6841 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6901 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6961 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7021 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7081 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7141 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7201 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7261 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7321 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7381 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7441 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7501 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7561 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7621 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7681 tggat
//
