LOCUS       sequence-483912-        5987 bp    DNA     circular SYN 20-AUG-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 18 to 52)
  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 5987)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 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: SAGA003
FEATURES             Location/Qualifiers
     source          1..5987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..52
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             64..75
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             120..128
                     /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             135..848
                     /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     788..808
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             858..869
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             979..2169
                     /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(1331..1350)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2765..2784
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2851..2953)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2981..3075)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            3251..3359
                     /note="incP origin of transfer"
     CDS             complement(3420..4568)
                     /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      4961..5492
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5895..5981)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaagc gttgcgcttg acggctagct cagtcctagg tacagtgcta gctactagag
       61 attaaagagg agaaactata tatgaaagca attttcgtac tgaaacatct taatcatgct
      121 aaggaggttt tctaatgagc aaaggagaag aacttttcac tggagttgtg ccaattcttg
      181 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
      241 atgctacata cggaaaactc acccttaaat ttatttgcac tactggaaaa ctacctgttc
      301 catggccaac acttgtcact actctgacct atggcgttca atgcttttcc cgttatccgg
      361 atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac
      421 gcactatatc tttcaaagat gacgggaact acaagacgcg tgctgaagtc aagtttgaag
      481 gtgataccct tgttaatcgt atcgagttaa agggtattga ttttaaagaa gatggaaaca
      541 ttctcggaca caaactagag tacaactata actcacacaa tgtatacatc acggcagaca
      601 aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacattgaa gatggttccg
      661 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
      721 cagacaacca ttacctgtcg acacaatctg ccctttcgaa agatcccaac gaaaagcgtg
      781 accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgagc
      841 tctacaaatg accaattatt gaaggccgct aacgcggcct ttttttgttt ctggtctccc
      901 aaaaatttat ttgcttatta atcatccggc tcgtataatg tgtggagggc ccaagttcac
      961 ttaaaaagga gagcgacgat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc
     1021 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat
     1081 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg
     1141 atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca
     1201 gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc
     1261 gtcctgtgga ttctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1321 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1381 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1441 ggcgccatct ccctgcacgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1501 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgccgtcc gatgcccttg
     1561 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgaccat tgtggccgca
     1621 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     1681 attttcggcg aggaccgctt tcgctggagc gcgacgatga gcggcctgtc gcttgcggta
     1741 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     1801 ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg
     1861 gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc
     1921 atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga
     1981 cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc
     2041 gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc
     2101 gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc
     2161 tcgacctgag acaattgtcc agataaaaaa aatccttagc tttcgctaag gatgatttct
     2221 ttattgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     2281 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     2341 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     2401 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     2461 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     2521 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     2581 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     2641 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     2701 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     2761 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     2821 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     2881 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     2941 tacgtgccga tcaacgtctc attttcgcca actagtctgg attctcacca ataaaaaacg
     3001 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     3061 ctatcaacag gagtccaagg ggtccccaat aattacgatt taaatgacaa gggtcctttt
     3121 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt
     3181 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac
     3241 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca
     3301 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt
     3361 aggccggccg cgatgcaggt ggctgctgaa cccccagccg gaactgaccc cacaaggccc
     3421 tagcgtttgc aatgcaccag gtcatcattg acccaggcgt gttccaccag gccgctgcct
     3481 cgcaactctt cgcaggcttc gccaacctgc tcgcgccact tcttcacgcg ggtggaatcc
     3541 gatccgcaca tgaggcggaa ggtttccagc ttgagcgggt acggctcccg gtgcgagctg
     3601 aaatagtcga acatccgtcg ggccgtcggc gacagcttgc ggtacttctc ccatatgaat
     3661 ttcgtgtagt ggtcgccagc aaacagcacg acgatttcct cgtcgatcag gacctggcaa
     3721 cgggacgttt tcttgccacg gtccaggacg cggaagcggt gcagcagcga caccgattcc
     3781 aggtgcccaa cgcggtcgga cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat
     3841 tcctcggcct tcgtgtaata ccggccattg atcgaccagc ccaggtcctg gcaaagctcg
     3901 tagaacgtga aggtgatcgg ctcgccgata ggggtgcgct tcgcgtactc caacacctgc
     3961 tgccacacca gttcgtcatc gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg
     4021 tccttgttga cgtggaaaat gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg
     4081 cgcgtggtga acagggcaga gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc
     4141 cacggcgcaa tatcgaacaa ggaaagctgc atttccttga tctgctgctt cgtgtgtttc
     4201 agcaacgcgg cctgcttggc ttcgctgacc tgttttgcca ggtcctcgcc ggcggttttt
     4261 cgcttcttgg tcgtcatagt tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc
     4321 gcctcctgtt cgagacgacg cgaacgctcc acggcggccg atggcgcggg cagggcaggg
     4381 ggagccagtt gcacgctgtc gcgctcgatc ttggccgtag cttgctggac tatcgagccg
     4441 acggactgga aggtttcgcg gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca
     4501 tcctcggcgg aaaaccccgc gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc
     4561 cgattcattc accctccttg cgggattgcc ccggaattaa ttccccggat cgatccgtcg
     4621 atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     4681 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc
     4741 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     4801 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     4861 accgtttctg cggactggct ttctacgtgg ctgccatttt tggggtgagg ccgttcgcgg
     4921 ccgaggggcg cagcccctgg ggggatggga ggcccgcgtt agcgggccgg gagggttcga
     4981 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta
     5041 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg
     5101 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat
     5161 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc
     5221 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg
     5281 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag
     5341 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg
     5401 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag
     5461 ttttccgcga ggtatccaca acgccggcgg ccctacatgg ctctgctgta gtgagtgggt
     5521 tgcgctccgg cagcggtcct gatcccccgc agaaaaaaag gatctcaaga agatcctttg
     5581 atcttttcta cggcgcgccc aataccattg ttcattccac ggacaaaaac agagaaagga
     5641 aacgacagag gccaaaaagc ctcgctttca gcacctgtcg tttcctttct tttcagaggg
     5701 tattttaaat aaaaacatta agttatgacg aagaagaacg gaaacgcctt aaaccgaaaa
     5761 ttttcataaa tagcgaaaac ccgcgaggtc gccgccccgt aacctgtcgg atcaccggaa
     5821 aggacccgta aagtgataat gattatcatc tacatatcac aacgtgcgtg gattacgatt
     5881 ttctagggcg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
     5941 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcct
//
