LOCUS       sequence-347002-        7946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A helper plasmid for expression of the artificial T7ocr-gam-bet-exo 
            operon. The plasmid carries the chloramphenicol acetyltransferase 
            gene repressible by the CI repressor of lambda phage..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7946)
  AUTHORS   Bubnov DM, Yuzbashev TV, Khozov AA, Melkina OE, Vybornaya TV, Stan 
            GB, Sineoky SP
  TITLE     Robust counterselection and advanced lambdaRed recombineering enable
            markerless chromosomal integration of large heterologous constructs.
  JOURNAL   Nucleic Acids Res. 2022 Aug 26;50(15):8947-8960. doi: 
            10.1093/nar/gkac649.
  PUBMED    35920321
REFERENCE   2  (bases 1 to 7946)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-08-26- 26"; volume: "50"; issue: 
            "15"; pages: "8947-8960"
COMMENT     Sequence Label: pRedCmOcr
FEATURES             Location/Qualifiers
     source          1..7946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..119
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             883..1668
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             1665..2345
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      2346..2590
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      2751..2837
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2929..2956
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             3096..3755
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3162..3181)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        join(3793..3807,3808..3813,3814..3830,3831..3837)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3813..3829
                     /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)."
     protein_bind    3845..3861
                     /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)."
     RBS             3880..3891
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3910..3927
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3999..4016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4170..4758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4250..4269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4929..5720,5721..5789))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5552..5571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5790..5895)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      5921..6015
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6043..6891)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(6965..7801)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
ORIGIN
        1 caccacaatt cagcaaattg tgaacatcat cacgttcatc tttccctggt tgccaatggc
       61 ccattttcct gtcagtaacg agaaggtcgc gaattcaggc gctttttaga ctggtcgtaa
      121 tgaaatctct agggatccta ataactgcac gaggtaacac aagatggcta tgtctaacat
      181 gacttacaac aacgttttcg accacgctta cgaaatgctg aaagaaaaca tccgttatga
      241 tgacatccgt gacactgatg acctgcacga tgctattcac atggctgccg ataatgcagt
      301 tccgcactac tacgctgaca tctttagcgt aatggcaagt gagggcattg accttgagtt
      361 cgaagactct ggtctgatgc ctgacaccaa ggacgtaatc cgcatcctgc aagcgcgtat
      421 ctatgagcaa ttaacgattg acctctggga agacgcagaa gacttgctca atgaatactt
      481 ggaggaagtc gaggagtacg aggaggatga agagtaatct agactaatca gcccggcatt
      541 tcgcgggcga tattttcaca gctatttcag gagttcagcc atgaacgctt attacattca
      601 ggatcgtctt gaggctcaga gctgggcgcg tcactaccag cagctcgccc gtgaagagaa
      661 agaggcagaa ctggcagacg acatggaaaa aggcctgccc cagcacctgt ttgaatcgct
      721 atgcatcgat catttgcaac gccacggggc cagcaaaaaa tccattaccc gtgcgtttga
      781 tgacgatgtt gagtttcagg agcgcatggc agaacacatc cggtacatgg ttgaaaccat
      841 tgctcaccac caggttgata ttgattcaga ggtataaaac gaatgagtac tgcactcgca
      901 acgctggctg ggaagctggc tgaacgtgtc ggcatggatt ctgtcgaccc acaggaactg
      961 atcaccactc ttcgccagac ggcatttaaa ggtgatgcca gcgatgcgca gttcatcgca
     1021 ttactgatcg ttgccaacca gtacggcctt aatccgtgga cgaaagaaat ttacgccttt
     1081 cctgataagc agaatggcat cgttccggtg gtgggcgttg atggctggtc ccgcatcatc
     1141 aatgaaaacc agcagtttga tggcatggac tttgagcagg acaatgaatc ctgtacatgc
     1201 cggatttacc gcaaggaccg taatcatccg atctgcgtta ccgaatggat ggatgaatgc
     1261 cgccgcgaac cattcaaaac tcgcgaaggc agagaaatca cggggccgtg gcagtcgcat
     1321 cccaaacgga tgttacgtca taaagccatg attcagtgtg cccgtctggc cttcggattt
     1381 gctggtatct atgacaagga tgaagccgag cgcattgtcg aaaatactgc atacactgca
     1441 gaacgtcagc cggaacgcga catcactccg gttaacgatg aaaccatgca ggagattaac
     1501 actctgctga tcgccctgga taaaacatgg gatgacgact tattgccgct ctgttcccag
     1561 atatttcgcc gcgacattcg tgcatcgtca gaactgacac aggccgaagc agtaaaagct
     1621 cttggattcc tgaaacagaa agccgcagag cagaaggtgg cagcatgaca ccggacatta
     1681 tcctgcagcg taccgggatc gatgtgagag ctgtcgaaca gggggatgat gcgtggcaca
     1741 aattacggct cggcgtcatc accgcttcag aagttcacaa cgtgatagca aaaccccgct
     1801 ccggaaagaa gtggcctgac atgaaaatgt cctacttcca caccctgctt gctgaggttt
     1861 gcaccggtgt ggctccggaa gttaacgcta aagcactggc ctggggaaaa cagtacgaga
     1921 acgacgccag aaccctgttt gaattcactt ccggcgtgaa tgttactgaa tccccgatca
     1981 tctatcgcga cgaaagtatg cgtaccgcct gctctcccga tggtttatgc agtgacggca
     2041 acggccttga actgaaatgc ccgtttacct cccgggattt catgaagttc cggctcggtg
     2101 gtttcgaggc cataaagtca gcttacatgg cccaggtgca gtacagcatg tgggtgacgc
     2161 gaaaaaatgc ctggtacttt gccaactatg acccgcgtat gaagcgtgaa ggcctgcatt
     2221 atgtcgtgat tgagcgggat gaaaagtaca tggcgagttt tgacgagatc gtgccggagt
     2281 tcatcgaaaa aatggacgag gcactggctg aaattggttt tgtatttggg gagcaatggc
     2341 gatgacgcat cctcacgata atatccgggt aggcgcaatc actttcgtct actccgttac
     2401 aaagcgaggc tgggtatttc ccggcctttc tgttatccga aatccactga aagcacagcg
     2461 gctggctgag gagataaata ataaacgagg ggctgtatgc acaaagcatc ttctgttgag
     2521 ttaagaacga gtatcgagat ggcacatagc cttgctcaaa ttggaatcag gtttgtgcca
     2581 ataccagtag aaacagacga agaattctag gctggagaat ctcgatccgg actccctgac
     2641 gccacgccag gcgctggagt ggatctatcg gctgaaaagt ctggtgtaag gatcgtggca
     2701 gacaagcccg tcagggcgcg tccatgcgag agtagggaac tgccaggcat caaataaaac
     2761 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2821 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     2881 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc
     2941 tgacggatgg cctttttgcg tttctacaaa ctcttcctgc catgagataa ccatctgcgg
     3001 tgataaatta tctctggcgg tgttgaccta aataccactg gcggtgatac tgagcacatc
     3061 agcaggcgca ctgaccagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     3121 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     3181 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     3241 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     3301 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     3361 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     3421 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     3481 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     3541 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     3601 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     3661 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     3721 aacagtactg cgatgagtgg cagggcgggg cgtaacatgg ccgctaggcc ctttcgtctt
     3781 cacctcgaga aatcataaaa aatttatttg ctttgtgagc ggataacaat tataatagat
     3841 tcaattgtga gcggataaca atttcacaca gaattcatta aagaggagaa attaactatg
     3901 agaggatcgc atcaccatca ccatcacgga tctatgcggt gtgaaatacc gcacagatgc
     3961 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4021 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4081 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4141 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4201 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4261 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4321 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4381 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4441 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4501 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4561 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4621 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4681 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4741 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4801 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4861 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4921 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4981 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5041 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5101 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5161 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5221 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5281 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5341 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5401 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5461 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5521 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5581 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5641 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5701 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5761 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5821 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5881 atagggggtt ccgcgcacat ttccccgaaa agtgccacct gactcctgtt gatagatcca
     5941 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     6001 tttattggtg agaatgacgt ccgtcggcat cggcatggcg aattaatctt tctgcgaatt
     6061 gagatgacgc cactggctgg gcgtcatccc ggtttcccgg gtaaacacca ccgaaaaata
     6121 gttactatct tcaaagccac attcggtcga aatatcactg attaacaggc ggctatgctg
     6181 gagaagatat tgcgcatgac acactctgac ctgtcgcaga tattgattga tggtcattcc
     6241 agtctgctgg cgaaattgct gacgcaaaac gcgctcactg cacgatgcct catcacaaaa
     6301 tttatccagc gcaaagggac ttttcaggct agccgccagc cgggtaatca gcttatccag
     6361 caacgtttcg ctggatgttg gcggcaacga atcactggtg taacgatggc gattcagcaa
     6421 catcaccaac tgcccgaaca gcaactcagc catttcgtta gcaaacggca catgctgact
     6481 actttcatgc tcaagctgac cgataacctg ccgcgcctgc gccatcccca tgctacctaa
     6541 gcgccagtgt ggttgccctg cgctggcgtt aaatcccgga atcgccccct gccagtcaag
     6601 attcagcttc agacgctccg ggcaataaat aatattctgc aaaaccagat cgttaacgga
     6661 agcgtaggag tgtttatcgt cagcatgaat gtaaaagaga tcgccacggg taatgcgata
     6721 agggcgatcg ttgagtacat gcaggccatt accgcgccag acaatcacca gctcacaaaa
     6781 atcatgtgta tgttcagcaa agacatcttg cggataacgg tcagccacag cgactgcctg
     6841 ctggtcgctg gcaaaaaaat catctttgag aagttttaac tgatgcgcca ccgtggctac
     6901 ctcggccaga gaacgaagtt gattattcgc aatatggcgt acaaatacgt tgagaagatt
     6961 cgcgttattg cagaaagcca tcccgtccct ggcgaatatc acgcggtgac cagttaaact
     7021 ctcggcgaaa aagcgtcgaa aagtggttac tgtcgctgaa tccacagcga taggcgatgt
     7081 cagtaacgct ggcctcgctg tggcgtagca gatgtcgggc tttcatcagt cgcaggcggt
     7141 tcaggtatcg ctgaggcgtc agtcccgttt gctgcttaag ctgccgatgt agcgtacgca
     7201 gtgaaagaga aaattgatcc gccacggcat cccaattcac ctcatcggca aaatggtcct
     7261 ccagccaggc cagaagcaag ttgagacgtg atgcgctgtt ttccaggttc tcctgcaaac
     7321 tgcttttacg cagcaagagc agtaattgca taaacaagat ctcgcgactg gcggtcgagg
     7381 gtaaatcatt ttccccttcc tgctgttcca tctgtgcaac cagctgtcgc acctgctgca
     7441 atacgctgtg gttaacgcgc cagtgagacg gatactgccc atccagctct tgtggcagca
     7501 actgattcag cccggcgaga aactgaaatc gatccggcga gcgatacagc acattggtca
     7561 gacacagatt atcggtatgt tcatacagat gccgatcatg atcgcgtacg aaacagaccg
     7621 tgccaccggt gatggtatag ggctgcccat taaacacatg aatacccgtg ccatgttcga
     7681 caatcacaat ttcatgaaaa tcatgatgat gttcaggaaa atccgcctgc gggagccggg
     7741 gttctatcgc cacggacgcg ttaccagacg gaaaaaaatc cacactatgt aatacggtca
     7801 tactggcctc ctgatgtcgt caacacggcg aaatagtaat cacgaggtca ggttcttacc
     7861 ttaaattttc gacggaaaac cacgtaaaaa acgtcgattt ttcaagatac agcgtgaatt
     7921 ttcaggaaat gcggtgagca tcacat
//
