LOCUS       sequence-171106-        4269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pK3FS-CYC1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4269)
  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 4269)
  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-CYC1
FEATURES             Location/Qualifiers
     source          1..4269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     365..382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     469..488
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        469..487
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(545..578)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            620..1975
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        620..963
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             964..1773
                     /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(1031..1050)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1779..1975
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(2315..2348)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2371..2442
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(2491..2510)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2610..2632
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2670..2688)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2756..2860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2861..2929,2930..3721)
                     /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(3079..3098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3892..4269,1..211)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
       61 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      121 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      181 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      241 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      301 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      361 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
      421 cgcgcgttgg ccgattcatt aatgcaggtt aacctggctt atcgaaatta atacgactca
      481 ctatagggag accggcagat ccgcggccgc gtacgctgca ggtcgacgga tctgatatca
      541 cctaataact tcgtatagca tacattatac gaagttatat taagggttct cgacgtcccc
      601 gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt
      661 gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt
      721 ataatcattt gcatccatac gttttgatgg ccgcacggcg cgaagcaaaa attacggctc
      781 ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca
      841 cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca
      901 tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca
      961 accatgggta aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct
     1021 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1081 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1141 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1201 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     1261 cccggcaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     1321 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     1381 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     1441 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     1501 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     1561 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     1621 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     1681 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     1741 cagtttcatt tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg
     1801 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     1861 atgttagcgt gatttatatt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     1921 cgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     1981 gattcgatac taacgccgcc atccggtgtc gagatctgcg ttggttggtg gatcaagccc
     2041 acgcgtaggc aatcctcgag cagatccgcc aggcgtgtat atatagcgtg gatggccagg
     2101 caactttagt gctgacacat acaggcatat atatatgtgt gcgacgacac atgatcatat
     2161 ggcatgcatg tgctctgtat gtatataaaa ctcttgtttt cttcttttct ctaaatattc
     2221 tttccttata cattaggacc tttgcagcat aaattactat acttctatag acacacaaac
     2281 acaaatacac acactaaatt aatatctaga cctaataact tcgtatagca tacattatac
     2341 gaagttatat taagggttgt cgacaccatg gactataaag acgacgacga caaagattat
     2401 aaggacgacg acgataagga ttacaaggac gacgacgata aaggatccat ggcgacagag
     2461 aattcatcga tgctcgaggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2521 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2581 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2641 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2701 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2821 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2881 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2941 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3001 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3061 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3121 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3181 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3241 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3301 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3361 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3421 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3481 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3541 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3601 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3661 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3721 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3781 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3841 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3901 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3961 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4021 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4081 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4141 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4201 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4261 actgagata
//
