LOCUS       sequence-484013-        7391 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 7391)
  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 7391)
  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: SAGA032
FEATURES             Location/Qualifiers
     source          1..7391
                     /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
     primer_bind     1209..1228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1209..1227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1232..1256
                     /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             1268..1279
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             1324..1332
                     /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             1339..2052
                     /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     1992..2012
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2122..2168
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2218..2236)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2232..2279
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2383..3573
                     /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(2735..2754)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4169..4188
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4255..4357)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(4415..4509)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            4655..4763
                     /note="incP origin of transfer"
     CDS             complement(4824..5972)
                     /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      6365..6896
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(7299..7385)
                     /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 cgcgaaatta atacgactca ctatagggag aggaattgtg agcggataac aattcctact
     1261 agagattaaa gaggagaaac tatatatgaa agcaattttc gtactgaaac atcttaatca
     1321 tgctaaggag gttttctaat gagcaaagga gaagaacttt tcactggagt tgtgccaatt
     1381 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
     1441 ggtgatgcta catacggaaa actcaccctt aaatttattt gcactactgg aaaactacct
     1501 gttccatggc caacacttgt cactactctg acctatggcg ttcaatgctt ttcccgttat
     1561 ccggatcaca tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag
     1621 gaacgcacta tatctttcaa agatgacggg aactacaaga cgcgtgctga agtcaagttt
     1681 gaaggtgata cccttgttaa tcgtatcgag ttaaagggta ttgattttaa agaagatgga
     1741 aacattctcg gacacaaact agagtacaac tataactcac acaatgtata catcacggca
     1801 gacaaacaaa agaatggaat caaagctaac ttcaaaattc gccacaacat tgaagatggt
     1861 tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt
     1921 ttaccagaca accattacct gtcgacacaa tctgcccttt cgaaagatcc caacgaaaag
     1981 cgtgaccaca tggtccttct tgagtttgta actgctgctg ggattacaca tggcatggat
     2041 gagctctaca aatgatcgac ctagcataac cccgcggggc ctcttcgggg gtctcgcggg
     2101 gttttttgct gaaagaagct tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     2161 cgttttatct gttgtttgtc gccaaagccc gaaaggaagc tgagttggct gctgccaccg
     2221 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2281 tgaaaggagg aactatatcc ggataaaaat ttatttgctt attaatcatc cggctcgtat
     2341 aatgtgtgga gggcccaagt tcacttaaaa aggagagcga cgatgaaatc taacaatgcg
     2401 ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag gcataggctt ggttatgccg
     2461 gtactgccgg gcctcttgcg ggatatcgtc cattccgaca gcatcgccag tcactatggc
     2521 gtgctgctag cgctatatgc gttgatgcaa tttctatgcg cacccgttct cggagcactg
     2581 tccgaccgct ttggccgccg cccagtcctg ctcgcttcgc tacttggagc cactatcgac
     2641 tacgcgatca tggcgaccac acccgtcctg tggattctct acgccggacg catcgtggcc
     2701 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     2761 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     2821 ggccccgtgg ccgggggact gttgggcgcc atctccctgc acgcaccatt ccttgcggcg
     2881 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     2941 ggagagcgcc gtccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3001 cggggcatga ccattgtggc cgcacttatg actgtcttct ttatcatgca actcgtagga
     3061 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     3121 atgagcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     3181 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     3241 gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc cttccccatt
     3301 atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat gctgtccagg
     3361 caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct taccagccta
     3421 acttcgatca ctggaccgct gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg
     3481 aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt
     3541 cgcggtgcat ggagccgggc cacctcgacc tgagacaatt gtccagataa aaaaaatcct
     3601 tagctttcgc taaggatgat ttctttattg taattcatta agcattctgc cgacatggaa
     3661 gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg
     3721 cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt
     3781 taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat
     3841 aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat
     3901 gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt
     3961 ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc
     4021 tttcattgcc atacgaaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa
     4081 ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag
     4141 ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt
     4201 acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc
     4261 ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc
     4321 attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccagaccct
     4381 tgtcatttaa atcgtaatta ttggggaccc ctggattctc accaataaaa aacgcccggc
     4441 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca
     4501 acaggagtcc aagactagtc ttttccgctg cataaccctg cttcggggtc attatagcga
     4561 ttttttcggt atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg
     4621 tagactttcc ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac
     4681 ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga
     4741 atcctgctct gcgaggctgg ccgtaggccg gccgcgatgc aggtggctgc tgaaccccca
     4801 gccggaactg accccacaag gccctagcgt ttgcaatgca ccaggtcatc attgacccag
     4861 gcgtgttcca ccaggccgct gcctcgcaac tcttcgcagg cttcgccaac ctgctcgcgc
     4921 cacttcttca cgcgggtgga atccgatccg cacatgaggc ggaaggtttc cagcttgagc
     4981 gggtacggct cccggtgcga gctgaaatag tcgaacatcc gtcgggccgt cggcgacagc
     5041 ttgcggtact tctcccatat gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt
     5101 tcctcgtcga tcaggacctg gcaacgggac gttttcttgc cacggtccag gacgcggaag
     5161 cggtgcagca gcgacaccga ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc
     5221 gtcgcctgta ggcgcgacag gcattcctcg gccttcgtgt aataccggcc attgatcgac
     5281 cagcccaggt cctggcaaag ctcgtagaac gtgaaggtga tcggctcgcc gataggggtg
     5341 cgcttcgcgt actccaacac ctgctgccac accagttcgt catcgtcggc ccgcagctcg
     5401 acgccggtgt aggtgatctt cacgtccttg ttgacgtgga aaatgacctt gttttgcagc
     5461 gcctcgcgcg ggattttctt gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt
     5521 ggcatcgctc gcatcgtgtc cggccacggc gcaatatcga acaaggaaag ctgcatttcc
     5581 ttgatctgct gcttcgtgtg tttcagcaac gcggcctgct tggcttcgct gacctgtttt
     5641 gccaggtcct cgccggcggt ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg
     5701 gtcatcgact tcgccaaacc tgccgcctcc tgttcgagac gacgcgaacg ctccacggcg
     5761 gccgatggcg cgggcagggc agggggagcc agttgcacgc tgtcgcgctc gatcttggcc
     5821 gtagcttgct ggactatcga gccgacggac tggaaggttt cgcggggcgc acgcatgacg
     5881 gtgcggcttg cgatggtttc ggcatcctcg gcggaaaacc ccgcgtcgat cagttcttgc
     5941 ctgtatgcct tccggtcaaa cgtccgattc attcaccctc cttgcgggat tgccccggaa
     6001 ttaattcccc ggatcgatcc gtcgatcttg atcccctgcg ccatcagatc cttggcggca
     6061 agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg
     6121 gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc
     6181 cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt
     6241 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtggctgcca
     6301 tttttggggt gaggccgttc gcggccgagg ggcgcagccc ctggggggat gggaggcccg
     6361 cgttagcggg ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt gcgcggtcac
     6421 gcgcacaggg cgcagccctg gttaaaaaca aggtttataa atattggttt aaaagcaggt
     6481 taaaagacag gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat gctggatttt
     6541 ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc agcactctgc
     6601 ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct caagtgtcaa
     6661 taccgcaggg cacttatccc caggcttgtc cacatcatct gtgggaaact cgcgtaaaat
     6721 caggcgtttt cgccgatttg cgaggctggc cagctccacg tcgccggccg aaatcgagcc
     6781 tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc aacgtccgcc
     6841 cctcatctgt cagtgagggc caagttttcc gcgaggtatc cacaacgccg gcggccctac
     6901 atggctctgc tgtagtgagt gggttgcgct ccggcagcgg tcctgatccc ccgcagaaaa
     6961 aaaggatctc aagaagatcc tttgatcttt tctacggcgc gcccaatacc attgttcatt
     7021 ccacggacaa aaacagagaa aggaaacgac agaggccaaa aagcctcgct ttcagcacct
     7081 gtcgtttcct ttcttttcag agggtatttt aaataaaaac attaagttat gacgaagaag
     7141 aacggaaacg ccttaaaccg aaaattttca taaatagcga aaacccgcga ggtcgccgcc
     7201 ccgtaacctg tcggatcacc ggaaaggacc cgtaaagtga taatgattat catctacata
     7261 tcacaacgtg cgtggattac gattttctag ggcggcggat ttgtcctact caggagagcg
     7321 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     7381 atttgatgcc t
//
