LOCUS       sequence-484214-        7448 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Plasmid contains the triple selection cassette gfp -RK2 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 7448)
  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 7448)
  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: SAGA040
FEATURES             Location/Qualifiers
     source          1..7448
                     /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             1271..1293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1312..1329
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1339..1356
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1360..1392
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1396..2109
                     /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     2049..2069
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2179..2225
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2275..2293)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2289..2336
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2440..3630
                     /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(2792..2811)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4226..4245
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4312..4414)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(4472..4566)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            4712..4820
                     /note="incP origin of transfer"
     CDS             complement(4881..6029)
                     /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      6422..6953
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(7356..7442)
                     /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 aattcccctc
     1261 tagaaataat tttgtttaac tttaagaagg agaatccgga tgggtagcag ccatcatcat
     1321 catcatcaca gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga
     1381 cagcaaatgg gtcgcatgag caaaggagaa gaacttttca ctggagttgt gccaattctt
     1441 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt
     1501 gatgctacat acggaaaact cacccttaaa tttatttgca ctactggaaa actacctgtt
     1561 ccatggccaa cacttgtcac tactctgacc tatggcgttc aatgcttttc ccgttatccg
     1621 gatcacatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     1681 cgcactatat ctttcaaaga tgacgggaac tacaagacgc gtgctgaagt caagtttgaa
     1741 ggtgataccc ttgttaatcg tatcgagtta aagggtattg attttaaaga agatggaaac
     1801 attctcggac acaaactaga gtacaactat aactcacaca atgtatacat cacggcagac
     1861 aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacattga agatggttcc
     1921 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     1981 ccagacaacc attacctgtc gacacaatct gccctttcga aagatcccaa cgaaaagcgt
     2041 gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag
     2101 ctctacaaat gatcgaccta gcataacccc gcggggcctc ttcgggggtc tcgcggggtt
     2161 ttttgctgaa agaagcttca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     2221 tttatctgtt gtttgtcgcc aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2281 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2341 aaggaggaac tatatccgga taaaaattta tttgcttatt aatcatccgg ctcgtataat
     2401 gtgtggaggg cccaagttca cttaaaaagg agagcgacga tgaaatctaa caatgcgctc
     2461 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
     2521 ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg
     2581 ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc
     2641 gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac
     2701 gcgatcatgg cgaccacacc cgtcctgtgg attctctacg ccggacgcat cgtggccggc
     2761 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     2821 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     2881 cccgtggccg ggggactgtt gggcgccatc tccctgcacg caccattcct tgcggcggcg
     2941 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     3001 gagcgccgtc cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     3061 ggcatgacca ttgtggccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     3121 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     3181 agcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     3241 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggccgac
     3301 gcgctgggct acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt ccccattatg
     3361 attcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct gtccaggcag
     3421 gtagatgacg accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact
     3481 tcgatcactg gaccgctgat cgtcacggcg atttatgccg cctcggcgag cacatggaac
     3541 gggttggcat ggattgtagg cgccgcccta taccttgtct gcctccccgc gttgcgtcgc
     3601 ggtgcatgga gccgggccac ctcgacctga gacaattgtc cagataaaaa aaatccttag
     3661 ctttcgctaa ggatgatttc tttattgtaa ttcattaagc attctgccga catggaagcc
     3721 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     3781 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     3841 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     3901 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     3961 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     4021 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     4081 cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     4141 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     4201 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4261 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4321 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4381 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agacccttgt
     4441 catttaaatc gtaattattg gggacccctg gattctcacc aataaaaaac gcccggcggc
     4501 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     4561 ggagtccaag actagtcttt tccgctgcat aaccctgctt cggggtcatt atagcgattt
     4621 tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag
     4681 actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg
     4741 cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc
     4801 ctgctctgcg aggctggccg taggccggcc gcgatgcagg tggctgctga acccccagcc
     4861 ggaactgacc ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt gacccaggcg
     4921 tgttccacca ggccgctgcc tcgcaactct tcgcaggctt cgccaacctg ctcgcgccac
     4981 ttcttcacgc gggtggaatc cgatccgcac atgaggcgga aggtttccag cttgagcggg
     5041 tacggctccc ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg
     5101 cggtacttct cccatatgaa tttcgtgtag tggtcgccag caaacagcac gacgatttcc
     5161 tcgtcgatca ggacctggca acgggacgtt ttcttgccac ggtccaggac gcggaagcgg
     5221 tgcagcagcg acaccgattc caggtgccca acgcggtcgg acgtgaagcc catcgccgtc
     5281 gcctgtaggc gcgacaggca ttcctcggcc ttcgtgtaat accggccatt gatcgaccag
     5341 cccaggtcct ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc
     5401 ttcgcgtact ccaacacctg ctgccacacc agttcgtcat cgtcggcccg cagctcgacg
     5461 ccggtgtagg tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc
     5521 tcgcgcggga ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc
     5581 atcgctcgca tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg catttccttg
     5641 atctgctgct tcgtgtgttt cagcaacgcg gcctgcttgg cttcgctgac ctgttttgcc
     5701 aggtcctcgc cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc
     5761 atcgacttcg ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc
     5821 gatggcgcgg gcagggcagg gggagccagt tgcacgctgt cgcgctcgat cttggccgta
     5881 gcttgctgga ctatcgagcc gacggactgg aaggtttcgc ggggcgcacg catgacggtg
     5941 cggcttgcga tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg
     6001 tatgccttcc ggtcaaacgt ccgattcatt caccctcctt gcgggattgc cccggaatta
     6061 attccccgga tcgatccgtc gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     6121 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     6181 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac
     6241 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc
     6301 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg gctgccattt
     6361 ttggggtgag gccgttcgcg gccgaggggc gcagcccctg gggggatggg aggcccgcgt
     6421 tagcgggccg ggagggttcg agaagggggg gcacccccct tcggcgtgcg cggtcacgcg
     6481 cacagggcgc agccctggtt aaaaacaagg tttataaata ttggtttaaa agcaggttaa
     6541 aagacaggtt agcggtggcc gaaaaacggg cggaaaccct tgcaaatgct ggattttctg
     6601 cctgtggaca gcccctcaaa tgtcaatagg tgcgcccctc atctgtcagc actctgcccc
     6661 tcaagtgtca aggatcgcgc ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac
     6721 cgcagggcac ttatccccag gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag
     6781 gcgttttcgc cgatttgcga ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc
     6841 ccctcatctg tcaacgccgc gccgggtgag tcggcccctc aagtgtcaac gtccgcccct
     6901 catctgtcag tgagggccaa gttttccgcg aggtatccac aacgccggcg gccctacatg
     6961 gctctgctgt agtgagtggg ttgcgctccg gcagcggtcc tgatcccccg cagaaaaaaa
     7021 ggatctcaag aagatccttt gatcttttct acggcgcgcc caataccatt gttcattcca
     7081 cggacaaaaa cagagaaagg aaacgacaga ggccaaaaag cctcgctttc agcacctgtc
     7141 gtttcctttc ttttcagagg gtattttaaa taaaaacatt aagttatgac gaagaagaac
     7201 ggaaacgcct taaaccgaaa attttcataa atagcgaaaa cccgcgaggt cgccgccccg
     7261 taacctgtcg gatcaccgga aaggacccgt aaagtgataa tgattatcat ctacatatca
     7321 caacgtgcgt ggattacgat tttctagggc ggcggatttg tcctactcag gagagcgttc
     7381 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     7441 tgatgcct
//
