LOCUS       sequence-488086-        2959 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 2959)
  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  JOURNAL   Unpublished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 2959)
  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            "Unpublished"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_FRTampFRT
FEATURES             Location/Qualifiers
     source          1..2959
                     /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        121..225
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(226..294,295..1086)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(444..463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    1099..1146
                     /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(1237..1255)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1238..1255)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1273..1289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1273..1289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      1360..1432
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1488..1844
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1958..2617)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2532..2551
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2618..2720)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2739..2766)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     2907..2924
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2908..2924
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2931..2950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2931..2949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggtctcagga gattccgggg atccgtcgac ctgcagttcg aagttcctat tctctagaaa
       61 gtataggaac ttcagagcgc ttttgaagct cacgcaggtg gcacttttcg gggaaatgtg
      121 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      181 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      241 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      301 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      361 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
      421 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
      481 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      541 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      601 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      661 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
      721 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
      781 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
      841 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
      901 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttctcgcgg tatcattgca
      961 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1021 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1081 tggtaataag gggatcttga agttcctatt ccgaagttcc tattctctag aaagtatagg
     1141 aacttcgaag cagctccagc ctacacgctg tcaggaggat cgctctgcca tctagcacgc
     1201 tgtcatgaga ccggatcctg acttgtattc cgcggttcta tagtgtcacc taaatagctt
     1261 ggcgtaattt aggtcatagc tgtttcctgt cggaccaaaa cgaaaaaagg cccccctttc
     1321 gggaggcctc ttttctggaa tttggtaccg agcaggcatc aaataaaacg aaaggctcag
     1381 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg ttgaacgctc tctgacgtac
     1441 cgcggccgcg tagaggatct gttgatcagc agttcaacct gttgatagta cgtactaagc
     1501 tctcatgttt cacgtactaa gctctcatgt ttaacgtact aagctctcat gtttaacgaa
     1561 ctaaaccctc atggctaacg tactaagctc tcatggctaa cgtactaagc tctcatgttt
     1621 cacgtactaa gctctcatgt ttgaacaata aaattaatat aaatcagcaa cttaaatagc
     1681 ctctaaggtt ttaagtttta taagaaaaaa aagaatatat aaggctttta aagcttttaa
     1741 ggtttaacgg ttgtggacaa caagccaggg atgtaacgca ctgagaagcc cttagagcct
     1801 ctcaaagcaa ttttgagtga cacaggaaca cttaacggct gacatgtgat catgtttgac
     1861 agcttatcat cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac
     1921 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcgt
     1981 cgcaatactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat
     2041 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtatag tatttgccca
     2101 tcgtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
     2161 aactcaccca gggattggct gacacgaaaa acatattctc aataaaccct ttagggaaat
     2221 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
     2281 aatcgtcgtg gtattcactc cagaggcttg aaaacgtttc agtttgctca tggaaaacgg
     2341 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa
     2401 attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
     2461 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagttgaacg gtctggttat
     2521 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
     2581 tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa
     2641 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     2701 aacctcttac gtgccgatca taacattgta gaaacgcaaa aaggccatcc gtcaggatgg
     2761 ccttctgctt aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctttca
     2821 gccaaaaaac ttaagaccgc cggtcttgtc cactaccttg cagtaatgcg gtggacagga
     2881 tcggcggttt tcttttctct tctcaatgta aaacgacggc cagtgaattg taatacgact
     2941 cactataggg ttcggatcc
//
