LOCUS       sequence-347001-        7556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LambdaRed-expressing plasmid carrying 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 7556)
  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 7556)
  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: pRedCm
FEATURES             Location/Qualifiers
     source          1..7556
                     /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             493..1278
                     /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             1275..1955
                     /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      1956..2200
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      2361..2447
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2539..2566
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             2706..3365
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2772..2791)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        join(3403..3417,3418..3423,3424..3440,3441..3447)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3423..3439
                     /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    3455..3471
                     /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             3490..3501
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3520..3537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3609..3626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3780..4368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3860..3879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4539..5330,5331..5399))
                     /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     5162..5181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5400..5505)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      5531..5625
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5653..6501)
                     /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(6575..7411)
                     /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 agactaatca gcccggcatt tcgcgggcga tattttcaca gctatttcag
      181 gagttcagcc atgaacgctt attacattca ggatcgtctt gaggctcaga gctgggcgcg
      241 tcactaccag cagctcgccc gtgaagagaa agaggcagaa ctggcagacg acatggaaaa
      301 aggcctgccc cagcacctgt ttgaatcgct atgcatcgat catttgcaac gccacggggc
      361 cagcaaaaaa tccattaccc gtgcgtttga tgacgatgtt gagtttcagg agcgcatggc
      421 agaacacatc cggtacatgg ttgaaaccat tgctcaccac caggttgata ttgattcaga
      481 ggtataaaac gaatgagtac tgcactcgca acgctggctg ggaagctggc tgaacgtgtc
      541 ggcatggatt ctgtcgaccc acaggaactg atcaccactc ttcgccagac ggcatttaaa
      601 ggtgatgcca gcgatgcgca gttcatcgca ttactgatcg ttgccaacca gtacggcctt
      661 aatccgtgga cgaaagaaat ttacgccttt cctgataagc agaatggcat cgttccggtg
      721 gtgggcgttg atggctggtc ccgcatcatc aatgaaaacc agcagtttga tggcatggac
      781 tttgagcagg acaatgaatc ctgtacatgc cggatttacc gcaaggaccg taatcatccg
      841 atctgcgtta ccgaatggat ggatgaatgc cgccgcgaac cattcaaaac tcgcgaaggc
      901 agagaaatca cggggccgtg gcagtcgcat cccaaacgga tgttacgtca taaagccatg
      961 attcagtgtg cccgtctggc cttcggattt gctggtatct atgacaagga tgaagccgag
     1021 cgcattgtcg aaaatactgc atacactgca gaacgtcagc cggaacgcga catcactccg
     1081 gttaacgatg aaaccatgca ggagattaac actctgctga tcgccctgga taaaacatgg
     1141 gatgacgact tattgccgct ctgttcccag atatttcgcc gcgacattcg tgcatcgtca
     1201 gaactgacac aggccgaagc agtaaaagct cttggattcc tgaaacagaa agccgcagag
     1261 cagaaggtgg cagcatgaca ccggacatta tcctgcagcg taccgggatc gatgtgagag
     1321 ctgtcgaaca gggggatgat gcgtggcaca aattacggct cggcgtcatc accgcttcag
     1381 aagttcacaa cgtgatagca aaaccccgct ccggaaagaa gtggcctgac atgaaaatgt
     1441 cctacttcca caccctgctt gctgaggttt gcaccggtgt ggctccggaa gttaacgcta
     1501 aagcactggc ctggggaaaa cagtacgaga acgacgccag aaccctgttt gaattcactt
     1561 ccggcgtgaa tgttactgaa tccccgatca tctatcgcga cgaaagtatg cgtaccgcct
     1621 gctctcccga tggtttatgc agtgacggca acggccttga actgaaatgc ccgtttacct
     1681 cccgggattt catgaagttc cggctcggtg gtttcgaggc cataaagtca gcttacatgg
     1741 cccaggtgca gtacagcatg tgggtgacgc gaaaaaatgc ctggtacttt gccaactatg
     1801 acccgcgtat gaagcgtgaa ggcctgcatt atgtcgtgat tgagcgggat gaaaagtaca
     1861 tggcgagttt tgacgagatc gtgccggagt tcatcgaaaa aatggacgag gcactggctg
     1921 aaattggttt tgtatttggg gagcaatggc gatgacgcat cctcacgata atatccgggt
     1981 aggcgcaatc actttcgtct actccgttac aaagcgaggc tgggtatttc ccggcctttc
     2041 tgttatccga aatccactga aagcacagcg gctggctgag gagataaata ataaacgagg
     2101 ggctgtatgc acaaagcatc ttctgttgag ttaagaacga gtatcgagat ggcacatagc
     2161 cttgctcaaa ttggaatcag gtttgtgcca ataccagtag aaacagacga agaattctag
     2221 gctggagaat ctcgatccgg actccctgac gccacgccag gcgctggagt ggatctatcg
     2281 gctgaaaagt ctggtgtaag gatcgtggca gacaagcccg tcagggcgcg tccatgcgag
     2341 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     2401 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     2461 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     2521 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     2581 ctcttcctgc catgagataa ccatctgcgg tgataaatta tctctggcgg tgttgaccta
     2641 aataccactg gcggtgatac tgagcacatc agcaggcgca ctgaccagga gctaaggaag
     2701 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta
     2761 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc
     2821 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct
     2881 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag
     2941 acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa
     3001 ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca
     3061 tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta
     3121 ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa
     3181 acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc
     3241 aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct
     3301 tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg
     3361 cgtaacatgg ccgctaggcc ctttcgtctt cacctcgaga aatcataaaa aatttatttg
     3421 ctttgtgagc ggataacaat tataatagat tcaattgtga gcggataaca atttcacaca
     3481 gaattcatta aagaggagaa attaactatg agaggatcgc atcaccatca ccatcacgga
     3541 tctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3601 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3661 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3721 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3781 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3841 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3901 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3961 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4021 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4081 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4141 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4201 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4261 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4321 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4381 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4441 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4501 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4561 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4621 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4681 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4741 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4801 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4861 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4921 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4981 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5041 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5101 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5161 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5221 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5281 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5341 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5401 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5461 atatttgaat gtatttagaa aaataaacaa atagggggtt ccgcgcacat ttccccgaaa
     5521 agtgccacct gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     5581 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatgacgt ccgtcggcat
     5641 cggcatggcg aattaatctt tctgcgaatt gagatgacgc cactggctgg gcgtcatccc
     5701 ggtttcccgg gtaaacacca ccgaaaaata gttactatct tcaaagccac attcggtcga
     5761 aatatcactg attaacaggc ggctatgctg gagaagatat tgcgcatgac acactctgac
     5821 ctgtcgcaga tattgattga tggtcattcc agtctgctgg cgaaattgct gacgcaaaac
     5881 gcgctcactg cacgatgcct catcacaaaa tttatccagc gcaaagggac ttttcaggct
     5941 agccgccagc cgggtaatca gcttatccag caacgtttcg ctggatgttg gcggcaacga
     6001 atcactggtg taacgatggc gattcagcaa catcaccaac tgcccgaaca gcaactcagc
     6061 catttcgtta gcaaacggca catgctgact actttcatgc tcaagctgac cgataacctg
     6121 ccgcgcctgc gccatcccca tgctacctaa gcgccagtgt ggttgccctg cgctggcgtt
     6181 aaatcccgga atcgccccct gccagtcaag attcagcttc agacgctccg ggcaataaat
     6241 aatattctgc aaaaccagat cgttaacgga agcgtaggag tgtttatcgt cagcatgaat
     6301 gtaaaagaga tcgccacggg taatgcgata agggcgatcg ttgagtacat gcaggccatt
     6361 accgcgccag acaatcacca gctcacaaaa atcatgtgta tgttcagcaa agacatcttg
     6421 cggataacgg tcagccacag cgactgcctg ctggtcgctg gcaaaaaaat catctttgag
     6481 aagttttaac tgatgcgcca ccgtggctac ctcggccaga gaacgaagtt gattattcgc
     6541 aatatggcgt acaaatacgt tgagaagatt cgcgttattg cagaaagcca tcccgtccct
     6601 ggcgaatatc acgcggtgac cagttaaact ctcggcgaaa aagcgtcgaa aagtggttac
     6661 tgtcgctgaa tccacagcga taggcgatgt cagtaacgct ggcctcgctg tggcgtagca
     6721 gatgtcgggc tttcatcagt cgcaggcggt tcaggtatcg ctgaggcgtc agtcccgttt
     6781 gctgcttaag ctgccgatgt agcgtacgca gtgaaagaga aaattgatcc gccacggcat
     6841 cccaattcac ctcatcggca aaatggtcct ccagccaggc cagaagcaag ttgagacgtg
     6901 atgcgctgtt ttccaggttc tcctgcaaac tgcttttacg cagcaagagc agtaattgca
     6961 taaacaagat ctcgcgactg gcggtcgagg gtaaatcatt ttccccttcc tgctgttcca
     7021 tctgtgcaac cagctgtcgc acctgctgca atacgctgtg gttaacgcgc cagtgagacg
     7081 gatactgccc atccagctct tgtggcagca actgattcag cccggcgaga aactgaaatc
     7141 gatccggcga gcgatacagc acattggtca gacacagatt atcggtatgt tcatacagat
     7201 gccgatcatg atcgcgtacg aaacagaccg tgccaccggt gatggtatag ggctgcccat
     7261 taaacacatg aatacccgtg ccatgttcga caatcacaat ttcatgaaaa tcatgatgat
     7321 gttcaggaaa atccgcctgc gggagccggg gttctatcgc cacggacgcg ttaccagacg
     7381 gaaaaaaatc cacactatgt aatacggtca tactggcctc ctgatgtcgt caacacggcg
     7441 aaatagtaat cacgaggtca ggttcttacc ttaaattttc gacggaaaac cacgtaaaaa
     7501 acgtcgattt ttcaagatac agcgtgaatt ttcaggaaat gcggtgagca tcacat
//
