LOCUS       sequence-356907-        7721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB Delta-Ring 3 (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7721)
  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 7721)
  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: pBEL2307
FEATURES             Location/Qualifiers
     source          1..7721
                     /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             5081..5098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5164..5182)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5178..5225
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5262..5717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5349..5368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5559..5580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5792..5825)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5904..6008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6009..6077,6078..6869)
                     /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(6227..6246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7040..7628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7529..7548
                     /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 gatctcggct 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 cgtgttctga cgctgaccct gaccgcgccg gaaagttacg gtattgatgc
     3661 gggtcagccg ctcattcttc acggcgtgca ggtaggccag gttatcgatc gtaaactcac
     3721 cagcaaaggc gtcaccttta ccgtcgccat cgagcctcag catcgcgaac tggtaaaagg
     3781 cgatagcaaa tttgtcgtca acagccgtgt cgatgtgaag gtggggctgg atggcgttga
     3841 gtttctcggt gccagcgcct cagaatggat caatggcggg atacgtattc tgccgggcga
     3901 taaaggtgag atgaaagcca gctatccact gtatgccaat ttggaaaaag cgctggagaa
     3961 cagccttagc gatttaccca ccacaaccgt gagtttgagt gcagagacgc tgccggatgt
     4021 gcaggcagga tcggtagtgc tgtaccgtaa atttgaagtt ggtgaagtga ttaccgtgcg
     4081 tccgcgagct aacgcgtttg atatcgatct gcatattaag ccggagtatc gcaaccttct
     4141 gaccagcaat agcgtgttct gggcagaagg cggggcgaaa gttcagctga atggtagtgg
     4201 tctgaccgta caggcatccc cgctctccag agcattaaag ggagccatta gcttcgacaa
     4261 cctcagcggt gccagcgcca gtcagcgtaa aggcgataag cgtattctgt atgcttccga
     4321 aacagcggcc cgtgcggttg gcgggcagat tacgcttcac gctttcgatg ccggaaaact
     4381 ggcggtcggg atgccaattc gctatctcgg tattgatatc gggcaaatcc agacgctgga
     4441 tctgattacc gcacgcaatg aagtacaggc aaaggcggta ctttacccgg aatatgtcca
     4501 gacctttgct cgcggtggta cgcgcttctc agtggtcaca ccgcaaattt cggcagctgg
     4561 cgttgagcat cttgatacta tcctccagcc gtatatcaac gtcgaaccag gccggggcaa
     4621 tcctcgccgc gactttgaat tacaagaggc caccattact gattcgcgtt acctggatgg
     4681 cttaagcatt attgttgaag cgccggaagc cggttcgtta ggcatcggta cgcctgtgct
     4741 gttccgtggt ctggaagtcg gtacggttac aggaatgacg ctggggacat tgtcagatcg
     4801 cgtgatgatt gcgatgcgca tcagtaaacg ctatcaacac ctggtgcgta acaattccgt
     4861 gttctggttg gcatcgggtt acagtctgga ctttggtctg acgggcggcg tagtgaaaac
     4921 cggcaccttt aaccagttta tccgtggcgg catcgccttc gccacgcctc cgggtacgcc
     4981 actggcaccg aaagcccagg aaggcaaaca cttcctgttg caggaaagtg aaccgaaaga
     5041 gtggcgtgaa tggggaactg cgcttcccaa aggcgagacc catcaccacc atcaccatta
     5101 aggccaggcc ggccgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     5161 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     5221 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     5281 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5341 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5401 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5461 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5521 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5581 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5641 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5701 attaacgctt acaatttagg tggcactttt cggggaacga cgacctgcag ccaagctagc
     5761 ttggctggac gtaaactcct cttcagacct aataacttcg tatagcatac attatacgaa
     5821 gttatattaa gggttattga atatgatcgg aattgggctg caggaattcg atagcttggc
     5881 tgcaggtcga cctcgaggat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5941 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6001 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6061 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6121 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6181 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6241 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6301 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6361 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6421 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6481 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6541 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6601 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6661 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6721 gtggctctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6781 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6841 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6901 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     7201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga t
//
