LOCUS       sequence-483926-        7975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the triple selection cassette gfp - tetARK2- 
            CmTrunc that allows for in vivo recombineering in E.coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7975)
  AUTHORS   Sepulchro AGV, Kozlowski HCJ, Norholm MHH
  TITLE     Simple and robust in vivo engineering of plasmid DNA at any copy 
            number in Escherichia coli.
  JOURNAL   Commun Biol. 2025 Jun 17;8(1):933. doi: 10.1038/s42003-025-08361-9.
  PUBMED    40527904
REFERENCE   2  (bases 1 to 7975)
  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: "Commun 
            Biol."; date: "2025-06-17"; volume: "8(1):933. doi"; pages: " 
            10.1038/s42003-025-08361-9"
COMMENT     Sequence Label: SAGA012
FEATURES             Location/Qualifiers
     source          1..7975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(18..866)
                     /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(940..1776)
                     /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"
     promoter        1922..2040
                     /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)"
     RBS             2052..2063
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2108..2116
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2123..2836
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     2776..2796
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2846..2857
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2967..4157
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMSGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(3319..3338)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4753..4772
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4839..4941)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(4999..5093)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            5239..5347
                     /note="incP origin of transfer"
     CDS             complement(5408..6556)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      6949..7480
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(7883..7969)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaagc gttgcgctta atctttctgc gaattgagat gacgccactg gctgggcgtc
       61 atcccggttt cccgggtaaa caccaccgaa aaatagttac tatcttcaaa gccacattcg
      121 gtcgaaatat cactgattaa caggcggcta tgctggagaa gatattgcgc atgacacact
      181 ctgacctgtc gcagatattg attgatggtc attccagtct gctggcgaaa ttgctgacgc
      241 aaaacgcgct cactgcacga tgcctcatca caaaatttat ccagcgcaaa gggacttttc
      301 aggctagccg ccagccgggt aatcagctta tccagcaacg tttcgctgga tgttggcggc
      361 aacgaatcac tggtgtaacg atggcgattc agcaacatca ccaactgccc gaacagcaac
      421 tcagccattt cgttagcaaa cggcacatgc tgactacttt catgctcaag ctgaccaata
      481 acctgccgcg cctgcgccat ccccatgcta cctaagcgcc agtgtggttg ccctgcgctg
      541 gcgttaaatc ccggaatcgc cccctgccag tcaagattca gcttcagacg ctccgggcaa
      601 taaataatat tctgcaaaac cagatcgtta acggaagcgt aggagtgttt atcatcagca
      661 tgaatgtaaa agagatcgcc acgggtaatg cgataagggc gatcgttgag tacatgcagg
      721 ccattaccgc gccagacaat caccagctca caaaaatcat gtgtatgttc agcaaagaca
      781 tcttgcggat aacggtcagc cacagcgact gcctgctggt cgctggcaaa aaaatcatct
      841 ttgagaagtt ttaactgatg cgccaccgtg gctacctcgg ccagagaacg aagttgatta
      901 ttcgcaatat ggcgtacaaa tacgttgaga agattcgcgt tattgcagaa agccatcccg
      961 tccctggcga atatcacgcg gtgaccagtt aaactctcgg cgaaaaagcg tcgaaaagtg
     1021 gttactgtcg ctgaatccac agcgataggc gatgtcagta acgctggcct cgctgtggcg
     1081 tagcagatgt cgggctttca tcagtcgcag gcggttcagg tatcgctgag gcgtcagtcc
     1141 cgtttgctgc ttaagctgcc gatgtagcgt acgcagtgaa agagaaaatt gatccgccac
     1201 ggcatcccaa ttcacctcat cggcaaaatg gtcctccagc caggccagaa gcaagttgag
     1261 acgtgatgcg ctgttttcca ggttctcctg caaactgctt ttacgcagca agagcagtaa
     1321 ttgcataaac aagatctcgc gactggcggt cgagggtaaa tcattttccc cttcctgctg
     1381 ttccatctgt gcaaccagct gtcgcacctg ctgcaatacg ctgtggttaa cgcgccagtg
     1441 agacggatac tgcccatcca gctcttgtgg cagcaactga ttcagcccgg cgagaaactg
     1501 aaatcgatcc ggcgagcgat acagcacatt ggtcagacac agattatcgg tatgttcata
     1561 cagatgccga tcatgatcgc gtacgaaaca gaccgtgcca ccggtgatgg tatagggctg
     1621 cccattaaac acatgaatac ccgtgccatg ttcgacaatc acaatttcat gaaaatcatg
     1681 atgatgttca ggaaaatccg cctgcgggag ccggggttct atcgccacgg acgcgttacc
     1741 agacggaaaa aaatccacac tatgtaatac ggtcatactg gcctcctgat gtcgtcaaca
     1801 cggcgaaata gtaatcacga ggtcaggttc ttaccttaaa ttttcgacgg aaaaccacgt
     1861 aaaaaacgtc gatttttcaa gatacagcgt gaattttcag gaaatgcggt gagcatcaca
     1921 tcaccacaat tcagcaaatt gtgaacatca tcacgttcat ctttccctgg ttgccaatgg
     1981 cccattttct tgtcagtaac gagaaggtcg cgaatccagg cgctttttag actggtcgta
     2041 tactagagat taaagaggag aaagggggga tgaaggctat tttcgtactg aaacatctta
     2101 atcatgctaa ggaggttttc taatgagcaa aggagaagaa cttttcactg gagttgtgcc
     2161 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
     2221 tgaaggtgat gctacatacg gaaaactcac ccttaaattt atttgcacta ctggaaaact
     2281 acctgttcca tggccaacac ttgtcactac tctgacctat ggcgttcaat gcttttcccg
     2341 ttatccggat cacatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt
     2401 acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtg ctgaagtcaa
     2461 gtttgaaggt gatacccttg ttaatcgtat cgagttaaag ggtattgatt ttaaagaaga
     2521 tggaaacatt ctcggacaca aactagagta caactataac tcacacaatg tatacatcac
     2581 ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccaca acattgaaga
     2641 tggttccgtt caactagcag accattatca acaaaatact ccaattggcg atggccctgt
     2701 ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaag atcccaacga
     2761 aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat
     2821 ggatgagctc tacaaatgac caattattga aggccgctaa cgcggccttt ttttgtttct
     2881 ggtctcccaa aaatttattt gcttattaat catccggctc gtataatgtg tggagggccc
     2941 aagttcactt aaaaaggaga gcgacgatga aatctaacaa tgcgctcatc gtcatcctcg
     3001 gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg ccgggcctct
     3061 tgcgggatat cgtccattcc gacagcatcg ccagtcacta tggcgtgctg ctagcgctat
     3121 atgcgttgat gcaatttcta tgcgcacccg ttctcggagc actgtccgac cgctttggcc
     3181 gccgcccagt cctgctcgct tcgctacttg gagccactat cgactacgcg atcatggcga
     3241 ccacacccgt cctgtggatt ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     3301 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     3361 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     3421 gactgttggg cgccatctcc ctgcacgcac cattccttgc ggcggcggtg ctcaacggcc
     3481 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgccgtccga
     3541 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgaccattg
     3601 tggccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     3661 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgagc ggcctgtcgc
     3721 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     3781 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg
     3841 tcttgctggc gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt
     3901 ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
     3961 atcagggaca gcttcaagga tcgctcgcgg ctcttaccag cctaacttcg atcactggac
     4021 cgctgatcgt cacggcgatt tatgccgcct cggcgagcac atggaacggg ttggcatgga
     4081 ttgtaggcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc
     4141 gggccacctc gacctgagac aattgtccag ataaaaaaaa tccttagctt tcgctaagga
     4201 tgatttcttt attgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     4261 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     4321 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     4381 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     4441 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     4501 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     4561 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacga
     4621 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     4681 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     4741 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     4801 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     4861 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     4921 gaacctctta cgtgccgatc aacgtctcat tttcgccaga cccttgtcat ttaaatcgta
     4981 attattgggg acccctggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     5041 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagact
     5101 agtcttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc
     5161 atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg
     5221 tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc
     5281 cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg
     5341 ctggccgtag gccggccgcg atgcaggtgg ctgctgaacc cccagccgga actgacccca
     5401 caaggcccta gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt tccaccaggc
     5461 cgctgcctcg caactcttcg caggcttcgc caacctgctc gcgccacttc ttcacgcggg
     5521 tggaatccga tccgcacatg aggcggaagg tttccagctt gagcgggtac ggctcccggt
     5581 gcgagctgaa atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg tacttctccc
     5641 atatgaattt cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg tcgatcagga
     5701 cctggcaacg ggacgttttc ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca
     5761 ccgattccag gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg
     5821 acaggcattc ctcggccttc gtgtaatacc ggccattgat cgaccagccc aggtcctggc
     5881 aaagctcgta gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca
     5941 acacctgctg ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga
     6001 tcttcacgtc cttgttgacg tggaaaatga ccttgttttg cagcgcctcg cgcgggattt
     6061 tcttgttgcg cgtggtgaac agggcagagc gggccgtgtc gtttggcatc gctcgcatcg
     6121 tgtccggcca cggcgcaata tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg
     6181 tgtgtttcag caacgcggcc tgcttggctt cgctgacctg ttttgccagg tcctcgccgg
     6241 cggtttttcg cttcttggtc gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca
     6301 aacctgccgc ctcctgttcg agacgacgcg aacgctccac ggcggccgat ggcgcgggca
     6361 gggcaggggg agccagttgc acgctgtcgc gctcgatctt ggccgtagct tgctggacta
     6421 tcgagccgac ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg
     6481 tttcggcatc ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat gccttccggt
     6541 caaacgtccg attcattcac cctccttgcg ggattgcccc ggaattaatt ccccggatcg
     6601 atccgtcgat cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt
     6661 tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct
     6721 tgctgtccat aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg
     6781 ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
     6841 gggtcagcac cgtttctgcg gactggcttt ctacgtggct gccatttttg gggtgaggcc
     6901 gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga
     6961 gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc
     7021 cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc
     7081 ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc
     7141 cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg
     7201 atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta
     7261 tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga
     7321 tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca
     7381 acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga
     7441 gggccaagtt ttccgcgagg tatccacaac gccggcggcc ctacatggct ctgctgtagt
     7501 gagtgggttg cgctccggca gcggtcctga tcccccgcag aaaaaaagga tctcaagaag
     7561 atcctttgat cttttctacg gcgcgcccaa taccattgtt cattccacgg acaaaaacag
     7621 agaaaggaaa cgacagaggc caaaaagcct cgctttcagc acctgtcgtt tcctttcttt
     7681 tcagagggta ttttaaataa aaacattaag ttatgacgaa gaagaacgga aacgccttaa
     7741 accgaaaatt ttcataaata gcgaaaaccc gcgaggtcgc cgccccgtaa cctgtcggat
     7801 caccggaaag gacccgtaaa gtgataatga ttatcatcta catatcacaa cgtgcgtgga
     7861 ttacgatttt ctagggcggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
     7921 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcct
//
