LOCUS       sequence-488088-        2829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenBraid2.0 Flp recombinase excisable antibiotic resistance 
            cassette in the grammar of an assembled transcription unit. Ready to
            be amplified to make gene knockouts..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2829)
  AUTHORS   Cooke MB, Welch KT, Deus Ramirez L, Wen AX, Marciano DC,, Herman C
  TITLE     GoldenBraid2.0 E. coli: A comprehensive and characterized toolkit 
            for enterics.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2829)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pR6Ko1_FRTspcFRT
FEATURES             Location/Qualifiers
     source          1..2829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    40..73
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        93..164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             165..956
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     protein_bind    969..1016
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(1107..1125)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1108..1125)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1143..1159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1143..1159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      1230..1302
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1358..1714
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1828..2487)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2402..2421
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2488..2590)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2609..2636)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     2777..2794
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2778..2794
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2801..2820
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2801..2819
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggtctcagga gattccgggg atccgtcgac ctgcagttcg aagttcctat tctctagaaa
       61 gtataggaac ttcagagcgc ttttgaagct cacgttcaaa tatgtatccg ctcatgagac
      121 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagg gaagcggtga
      181 tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac
      241 cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca
      301 gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt
      361 tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg
      421 tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg
      481 aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca
      541 cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg
      601 taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc
      661 taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg
      721 tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg
      781 atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg
      841 aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt
      901 tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataataag
      961 gggatcttga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcgaag
     1021 cagctccagc ctacacgctg tcaggaggat cgctctgcca tctagcacgc tgtcatgaga
     1081 ccggatcctg acttgtattc cgcggttcta tagtgtcacc taaatagctt ggcgtaattt
     1141 aggtcatagc tgtttcctgt cggaccaaaa cgaaaaaagg cccccctttc gggaggcctc
     1201 ttttctggaa tttggtaccg agcaggcatc aaataaaacg aaaggctcag tcgaaagact
     1261 gggcctttcg ttttatctgt tgtttgtcgg ttgaacgctc tctgacgtac cgcggccgcg
     1321 tagaggatct gttgatcagc agttcaacct gttgatagta cgtactaagc tctcatgttt
     1381 cacgtactaa gctctcatgt ttaacgtact aagctctcat gtttaacgaa ctaaaccctc
     1441 atggctaacg tactaagctc tcatggctaa cgtactaagc tctcatgttt cacgtactaa
     1501 gctctcatgt ttgaacaata aaattaatat aaatcagcaa cttaaatagc ctctaaggtt
     1561 ttaagtttta taagaaaaaa aagaatatat aaggctttta aagcttttaa ggtttaacgg
     1621 ttgtggacaa caagccaggg atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa
     1681 ttttgagtga cacaggaaca cttaacggct gacatgtgat catgtttgac agcttatcat
     1741 cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta
     1801 agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcgt cgcaatactg
     1861 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     1921 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtatag tatttgccca tcgtgaaaac
     1981 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     2041 gggattggct gacacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     2101 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     2161 gtattcactc cagaggcttg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     2221 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     2281 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     2341 ctttacggtc tttaaaaagg ccgtaatatc cagttgaacg gtctggttat aggtacattg
     2401 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     2461 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     2521 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     2581 gtgccgatca taacattgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     2641 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctttca gccaaaaaac
     2701 ttaagaccgc cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt
     2761 tcttttctct tctcaatgta aaacgacggc cagtgaattg taatacgact cactataggg
     2821 ttcggatcc
//
