LOCUS       sequence-484002-        7397 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 1 to 7397)
  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 7397)
  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: SAGA023
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(18..1100)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1062..1081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1101..1178)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(1209..1214,1215..1231,1232..1238)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1238..1262
                     /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             1274..1285
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             1330..1338
                     /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             1345..2058
                     /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     1998..2018
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2128..2174
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2224..2242)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2238..2285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2389..3579
                     /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(2741..2760)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4175..4194
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4261..4363)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(4421..4515)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            4661..4769
                     /note="incP origin of transfer"
     CDS             complement(4830..5978)
                     /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      6371..6902
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(7305..7391)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaagc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
       61 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
      121 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
      181 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
      241 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
      301 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
      361 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
      421 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
      481 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
      541 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
      601 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
      661 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
      721 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
      781 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
      841 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
      901 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
      961 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     1021 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     1081 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     1141 gccataccgc gaaaggtttt gcgccattcg atggtgtcgg atcgagatcg atctcgatcc
     1201 cgcgaaattt gacaattaat catccggctc gtataatgga attgtgagcg gataacaatt
     1261 cctactagag attaaagagg agaaactata tatgaaagca attttcgtac tgaaacatct
     1321 taatcatgct aaggaggttt tctaatgagc aaaggagaag aacttttcac tggagttgtg
     1381 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
     1441 ggtgaaggtg atgctacata cggaaaactc acccttaaat ttatttgcac tactggaaaa
     1501 ctacctgttc catggccaac acttgtcact actctgacct atggcgttca atgcttttcc
     1561 cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat
     1621 gtacaggaac gcactatatc tttcaaagat gacgggaact acaagacgcg tgctgaagtc
     1681 aagtttgaag gtgataccct tgttaatcgt atcgagttaa agggtattga ttttaaagaa
     1741 gatggaaaca ttctcggaca caaactagag tacaactata actcacacaa tgtatacatc
     1801 acggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacattgaa
     1861 gatggttccg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     1921 gtccttttac cagacaacca ttacctgtcg acacaatctg ccctttcgaa agatcccaac
     1981 gaaaagcgtg accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc
     2041 atggatgagc tctacaaatg atcgacctag cataaccccg cggggcctct tcgggggtct
     2101 cgcggggttt tttgctgaaa gaagcttcaa ataaaacgaa aggctcagtc gaaagactgg
     2161 gcctttcgtt ttatctgttg tttgtcgcca aagcccgaaa ggaagctgag ttggctgctg
     2221 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2281 ttttgctgaa aggaggaact atatccggat aaaaatttat ttgcttatta atcatccggc
     2341 tcgtataatg tgtggagggc ccaagttcac ttaaaaagga gagcgacgat gaaatctaac
     2401 aatgcgctca tcgtcatcct cggcaccgtc accctggatg ctgtaggcat aggcttggtt
     2461 atgccggtac tgccgggcct cttgcgggat atcgtccatt ccgacagcat cgccagtcac
     2521 tatggcgtgc tgctagcgct atatgcgttg atgcaatttc tatgcgcacc cgttctcgga
     2581 gcactgtccg accgctttgg ccgccgccca gtcctgctcg cttcgctact tggagccact
     2641 atcgactacg cgatcatggc gaccacaccc gtcctgtgga ttctctacgc cggacgcatc
     2701 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     2761 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     2821 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccctgcacgc accattcctt
     2881 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     2941 cataagggag agcgccgtcc gatgcccttg agagccttca acccagtcag ctccttccgg
     3001 tgggcgcggg gcatgaccat tgtggccgca cttatgactg tcttctttat catgcaactc
     3061 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     3121 gcgacgatga gcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     3181 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     3241 gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc
     3301 cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg
     3361 tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc
     3421 agcctaactt cgatcactgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc
     3481 acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg
     3541 ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgag acaattgtcc agataaaaaa
     3601 aatccttagc tttcgctaag gatgatttct ttattgtaat tcattaagca ttctgccgac
     3661 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     3721 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     3781 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     3841 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     3901 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     3961 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     4021 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
     4081 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     4141 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     4201 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     4261 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     4321 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
     4381 gacccttgtc atttaaatcg taattattgg ggacccctgg attctcacca ataaaaaacg
     4441 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     4501 ctatcaacag gagtccaaga ctagtctttt ccgctgcata accctgcttc ggggtcatta
     4561 tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg
     4621 ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
     4681 gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca
     4741 acgggaatcc tgctctgcga ggctggccgt aggccggccg cgatgcaggt ggctgctgaa
     4801 cccccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag gtcatcattg
     4861 acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc gccaacctgc
     4921 tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa ggtttccagc
     4981 ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg ggccgtcggc
     5041 gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc aaacagcacg
     5101 acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg gtccaggacg
     5161 cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga cgtgaagccc
     5221 atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata ccggccattg
     5281 atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg ctcgccgata
     5341 ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc gtcggcccgc
     5401 agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat gaccttgttt
     5461 tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga gcgggccgtg
     5521 tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa ggaaagctgc
     5581 atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc ttcgctgacc
     5641 tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt tcctcgcgtg
     5701 tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg cgaacgctcc
     5761 acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc gcgctcgatc
     5821 ttggccgtag cttgctggac tatcgagccg acggactgga aggtttcgcg gggcgcacgc
     5881 atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc gtcgatcagt
     5941 tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg cgggattgcc
     6001 ccggaattaa ttccccggat cgatccgtcg atcttgatcc cctgcgccat cagatccttg
     6061 gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc
     6121 cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg
     6181 taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag
     6241 cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgg
     6301 ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg cagcccctgg ggggatggga
     6361 ggcccgcgtt agcgggccgg gagggttcga gaaggggggg cacccccctt cggcgtgcgc
     6421 ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat tggtttaaaa
     6481 gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt gcaaatgctg
     6541 gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca tctgtcagca
     6601 ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag
     6661 tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg gaaactcgcg
     6721 taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc cggccgaaat
     6781 cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca agtgtcaacg
     6841 tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca acgccggcgg
     6901 ccctacatgg ctctgctgta gtgagtgggt tgcgctccgg cagcggtcct gatcccccgc
     6961 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggcgcgccc aataccattg
     7021 ttcattccac ggacaaaaac agaaaaagga aacgacagag gccaaaaagc ctcgctttca
     7081 gcacctgtcg tttcctttct tttcagaggg tattttaaat aaaaacatta agttatgacg
     7141 aagaagaacg gaaacgcctt aaaccgaaaa ttttcataaa tagcgaaaac ccgcgaggtc
     7201 gccgccccgt aacctgtcgg atcaccggaa aggacccgta aagtgataat gattatcatc
     7261 tacatatcac aacgtgcgtg gattacgatt ttctagggcg gcggatttgt cctactcagg
     7321 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt
     7381 cgttttattt gatgcct
//
