LOCUS       sequence-492175-        3638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenBraid2.0 Flp recombinase excisable antibiotic resistance 
            cassette in the grammar of an assembled transcription unit. May be 
            omega-assembled with another transcription unit..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3638)
  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  JOURNAL   Unpublished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 3638)
  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            "Unpublished"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: pBSKa2_FRTkanFRT
FEATURES             Location/Qualifiers
     source          1..3638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    44..77
                     /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)"
     CDS             445..1239
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(499..518)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1109..1128
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1252..1299
                     /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)."
     rep_origin      complement(1438..2026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1518..1537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2141..2243
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2244..3059
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2244..3059
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2317..2336)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3173..3628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3260..3279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3470..3491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgtctcagtc aggagattcc ggggatccgt cgacctgcag ttcgaagttc ctattctcta
       61 gaaagtatag gaacttcaga gcgcttttga agctcacgct gccgcaagca ctcagggcgc
      121 aagggctgct aaaggaagcg gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc
      181 cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag
      241 caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca
      301 agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta
      361 aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctgatcaa
      421 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
      481 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
      541 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
      601 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
      661 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
      721 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
      781 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
      841 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
      901 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
      961 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     1021 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     1081 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     1141 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     1201 cgcatcgcct tctatcgcct tcttgacgag ttcttctaat aaggggatct tgaagttcct
     1261 attccgaagt tcctattctc tagaaagtat aggaacttcg aagcagctcc agcctacacg
     1321 cttgagacga agcttgacac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1381 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1441 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1501 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1561 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1621 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1681 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1741 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1801 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1861 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1921 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1981 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2041 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2101 gagattatca aaaaggatct tcacctagat ccttttgtga tgatcggcac gtaagaggtt
     2161 ccaactttca ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat
     2221 tttcaggagc taaggaagct aaaatgagcc atattcaacg ggaaacgtct tgctcaaggc
     2281 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     2341 tcgggcaatc aggtgcgaca atctaccgat tgtatgggaa gcccgatgcg ccagagttgt
     2401 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     2461 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2521 atgcatggtt actcaccact gcgatcccag ggaaaacagc attccaggta ttagaagaat
     2581 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     2641 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgactc gctcaggcgc
     2701 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     2761 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     2821 tagtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     2881 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     2941 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     3001 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctgat
     3061 tttttttaag gcagttattg gtgcccttaa acgcctggtt gctacgcctg aataagtgat
     3121 aataagcgga tgaatggcag aaattcgatg ataagctgtc aaacatgatg ttacgcgccc
     3181 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3241 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3301 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3361 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccagcgccc
     3421 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3481 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3541 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3601 tttaacaaaa tattaacgct tacaatttta acaagctt
//
