LOCUS       sequence-171080-        4884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pK3F-PYK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4884)
  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 4884)
  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: pK3F-PYK1
FEATURES             Location/Qualifiers
     source          1..4884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            complement(599..1955)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(599..796)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(802..1611)
                     /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     1525..1544
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1612..1955)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    1997..2030
                     /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(2083..2102)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2084..2102)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2189..2206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2360..2948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2440..2459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3119..3910,3911..3979))
                     /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     3742..3761
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3980..4084)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4152..4170
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4208..4230)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4330..4349
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4430..4448
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4430..4447
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4486..4557)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     protein_bind    4580..4613
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gacactaaag gtacctagca tcatatggga aggaaaggaa atcacttgga agacatcaca
       61 agcattcatt taccaagaga aaaaatatgc attttagcta agatccattg aacaaagcac
      121 tcactcaact caactgaatg aacgaaagaa gaaagaacag tagaaaacac tttgtgacgg
      181 tgcggaacac atttacgtag ctatcatgct gaattctact atgaaaatct cccaatctgt
      241 cgatggcaaa acgacccacg tggcagagtt gggtcaagtg ccagtttctg gattaagtaa
      301 cagatacaga catcacacgc catagaggaa tcccgccgtt gcgagagatg gaaaacaata
      361 gagccgaaat tgtggaagcc cgatgtctgg gtgtacattt tttttttttc tttctttctc
      421 tttcaataat ctttcctttt tccatttagc ttgccggaaa aactttcggg tagcgaaaat
      481 ctttctgccg gaaaaattag ctattttttt cttccttatt atttttttag ttctgaagtt
      541 tgaccagggc gactagagat ctcgacactg gatggcggcg ttagtatcga atcgacagca
      601 gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc gcacttaact
      661 tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta acatttgatt
      721 aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg aaaacaagaa
      781 tctttttatt gtcagtactg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
      841 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
      901 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
      961 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     1021 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
     1081 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     1141 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     1201 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     1261 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttgc cggggatcgc
     1321 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     1381 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     1441 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     1501 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     1561 catgttggaa tttaatcgcg gcctcgaaac gtgagtcttt tccttaccca tggttgttta
     1621 tgttcggatg tgatgtgaga actgtatcct agcaagattt taaaaggaag tatatgaaag
     1681 aagaacctca gtggcaaatc ctaacctttt atatttctct acaggggcgc ggcgtgggga
     1741 caattcaacg cgtctgtgag gggagcgttt ccctgctcgc aggtctgcag cgaggagccg
     1801 taatttttgc ttcgcgccgt gcggccatca aaatgtatgg atgcaaatga ttatacatgg
     1861 ggatgtatgg gctaaatgta cgggcgacag tcacatcatg cccctgagct gcgcacgtca
     1921 agactgtcaa ggagggtatt ctgggcctcc atgtcgctgg ccgggtgacc cggcggggac
     1981 gtcgagaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtgatat
     2041 cagatccact agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat
     2101 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2161 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2221 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2281 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2341 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2401 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2461 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2521 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2581 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2641 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2701 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2761 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2821 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2881 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2941 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3001 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3061 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3121 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3181 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3241 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3301 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3361 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3421 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3481 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3541 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3601 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3661 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3721 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3781 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3841 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3901 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3961 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4021 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4081 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4141 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     4201 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4261 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     4321 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     4381 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     4441 actatagaac gcggccgcca ggatctcgcg gccgcccatg gatcctttat cgtcgtcgtc
     4501 cttgtaatcc ttatcgtcgt cgtccttata atctttgtcg tcgtcgtctt tatagtccat
     4561 ggtgtcgaca acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct
     4621 agatgtgatg atgttttatt tgttttgatt ggtgtcttgt aaatagaaac aagagagaat
     4681 aataaacaag ttaagaataa aaaaccaaag gatgaaaaag aatgaatatg aaaaagagta
     4741 gagaataact ttgaaagggg accatgatat aactggaaaa aagaggttct tggaaatgaa
     4801 aagttaccaa agagtattta taattcagaa aaaaaagcca acgaatatcg ttttgatggc
     4861 gagccttttt ttttttttta ggaa
//
