LOCUS       sequence-193147-        4797 bp    DNA     circular SYN 15-APR-2025
DEFINITION  N-ICE plasmid pK3FS-PYK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4797)
  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 4797)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-PYK1
FEATURES             Location/Qualifiers
     source          1..4797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1976..1994
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2032..2054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2154..2173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2222..2293)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     protein_bind    2316..2349
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(3216..4572)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(3216..3413)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3419..4228)
                     /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     4142..4161
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4229..4572)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    4614..4647
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4704..4723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4705..4723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     1981 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2161 agagcagatt gtactgagag tgcacctcga gcatcgatga attctctgtc gccatggatc
     2221 ctttatcgtc gtcgtccttg taatccttat cgtcgtcgtc cttataatct ttgtcgtcgt
     2281 cgtctttata gtccatggtg tcgacaaccc ttaatataac ttcgtataat gtatgctata
     2341 cgaagttatt aggtctagat gtgttgatgt tttatttgtt ttgattggtg tcttgtaaat
     2401 agaaacaaga gagaataata aacaagttaa gaataaaaaa ccaaaggatg aaaaagaatg
     2461 aatatgaaaa agagtagaga ataactttga aaggggacca tgatataact ggaaaaaaga
     2521 ggttcttgga aatgaaaagt taccaaagag tatttataat tcagaaaaaa aagccaacga
     2581 atatcgtttt gatggcgagc cttttttttt ttttaggaag acactaaagg tacctagcat
     2641 catatgggaa ggaaaggaaa tcacttggaa gacatcacaa gcattcattt accaagagaa
     2701 aaaatatgca ttttagctaa gatccattga acaaagcact cactcaactc aactgaatga
     2761 acgaaagaag aaagaacagt agaaaacact ttgtgacggt gcggaacaca tttacgtagc
     2821 tatcatgctg aattctacta tgaaaatctc ccaatctgtc gatggcaaaa cgacccacgt
     2881 ggcagagttg ggtcaagtgc cagtttctgg attaagtaac agatacagac atcacacgcc
     2941 atagaggaat cccgccgttg cgagagatgg aaaacaatag agcagaaatt gtggaagccc
     3001 gatgtctggg tgtacatttt ttttttcttt ctttctcttt caataatctt tcctttttcc
     3061 atttagcttg ccggaaaaac tttcgggtag cgaaaatctt tctgccggaa aaattagcta
     3121 tttttttctt ccttattatt tttttagttc tgaagtttga ccagggcgac tagagatctc
     3181 gacactggat ggcggcgtta gtatcgaatc gacagcagta tagcgaccag cattcacata
     3241 cgattgacgc atgatattac tttctgcgca cttaacttcg catctgggca gatgatgtcg
     3301 aggcgaaaaa aaatataaat cacgctaaca tttgattaaa atagaacaac tacaatataa
     3361 aaaaactata caaatgacaa gttcttgaaa acaagaatct ttttattgtc agtactgatt
     3421 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     3481 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     3541 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     3601 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     3661 aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc
     3721 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     3781 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     3841 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     3901 cttctaatac ctggaatgct gttttgccgg ggatcgcagt ggtgagtaac catgcatcat
     3961 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     4021 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     4081 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     4141 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     4201 tcgaaacgtg agtcttttcc ttacccatgg ttgtttatgt tcggatgtga tgtgagaact
     4261 gtatcctagc aagattttaa aaggaagtat atgaaagaag aacctcagtg gcaaatccta
     4321 accttttata tttctctaca ggggcgcggc gtggggacaa ttcaacgcgt ctgtgagggg
     4381 agcgtttccc tgctcgcagg tctgcagcga ggagccgtaa tttttgcttc gcgccgtgcg
     4441 gccatcaaaa tgtatggatg caaatgatta tacatgggga tgtatgggct aaatgtacgg
     4501 gcgacagtca catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg
     4561 ggcctccatg tcgctggccg ggtgacccgg cggggacgtc gagaaccctt aatataactt
     4621 cgtataatgt atgctatacg aagttattag gtgatatcag atccgtcgac ctgcagcgta
     4681 cgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     4741 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcg
//
