LOCUS       sequence-483932-        6862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the triple selection cassette gfp - tetApBR322-ROP-
            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 6862)
  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 6862)
  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: SAGA018
FEATURES             Location/Qualifiers
     source          1..6862
                     /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(4969..5063)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            5239..5347
                     /note="incP origin of transfer"
     rep_origin      5383..5971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5872..5891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6125..6142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6157..6297
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6223..6242)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6383..6405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6399..6590)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGNDGENL"
     terminator      complement(6770..6856)
                     /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 ttggacatga agtccaacaa 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 tttcgccaac tagtctggat tctcaccaat
     4981 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     5041 tactggatct atcaacagga gtccaagggg tccccaataa ttacgattta aatgacaagg
     5101 gtccttttcc 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 gccggccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5401 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5461 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5521 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5581 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5641 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5701 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5761 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5821 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg tagcttccag ggggaaacgc
     5881 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5941 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6001 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6061 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6121 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6181 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     6241 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     6301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatta
     6421 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag
     6481 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     6541 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     6601 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     6661 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     6721 ctggcggtat ggatgcggcg ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac
     6781 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag
     6841 cctttcgttt tatttgatgc ct
//
