LOCUS       sequence-171112-        3982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pK3FS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3982)
  AUTHORS   Zhang Y, Serratore ND, Briggs SD
  TITLE     N-ICE plasmids for generating N-terminal 3 x FLAG tagged genes that 
            allow Inducible, Constitutive, or Endogenous expression in 
            Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2016 Dec 12. doi: 10.1002/yea.3226.
  PUBMED    27943405
REFERENCE   2  (bases 1 to 3982)
  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: "Yeast. 2016
            Dec 12. doi: 10.1002/yea.3226."
COMMENT     Sequence Label: pK3FS
FEATURES             Location/Qualifiers
     source          1..3982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     490..509
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        490..508
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(566..599)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            641..1997
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        641..984
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             985..1794
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1052..1071)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1800..1997
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(2049..2082)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2105..2176
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(2225..2244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2344..2366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2404..2422)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2490..2594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2595..2663,2664..3455)
                     /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(2813..2832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3626..3982,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcaggt taacctggct
      481 tatcgaaatt aatacgactc actataggga gaccggcaga tccgcggccg cgtacgctgc
      541 aggtcgacgg atctgatatc acctaataac ttcgtatagc atacattata cgaagttata
      601 ttaagggttc tcgacgtccc cgccgggtca cccggccagc gacatggagg cccagaatac
      661 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
      721 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
      781 gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac
      841 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
      901 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca
      961 catcacatcc gaacataaac aaccatgggt aaggaaaaga ctcacgtttc gaggccgcga
     1021 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     1081 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     1141 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     1201 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     1261 tggttactca ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct
     1321 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     1381 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     1441 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     1501 gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc
     1561 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     1621 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     1681 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     1741 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagta
     1801 ctgacaataa aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg
     1861 tagttgttct attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca
     1921 tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa
     1981 tgctggtcgc tatactgctg tcgattcgat actaacgccg ccatccagtg tcgagatctc
     2041 tagacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg ttgtcgacac
     2101 catggactat aaagacgacg acgacaaaga ttataaggac gacgacgata aggattacaa
     2161 ggacgacgac gataaaggat ccatggcgac agagaattca tcgatgctcg aggtgcactc
     2221 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     2281 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2341 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     2401 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2461 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2821 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3121 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3961 cacagcccag cttggagcga ac
//
