LOCUS       sequence-169913-        5103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pH3F-PYK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5103)
  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 5103)
  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: pH3F-PYK1
FEATURES             Location/Qualifiers
     source          1..5103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            complement(818..2393)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(818..1015)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1021..2049)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1021..2049)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(2050..2393)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2435..2468
                     /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(2521..2540)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2522..2540)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2627..2644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2798..3386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2878..2897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3557..4348,4349..4417))
                     /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     4180..4199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4418..4522)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4590..4608
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4646..4668)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4768..4787
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4868..4886
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4868..4885
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4924..4995)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     protein_bind    5018..5051
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gaaacaagag agaataataa acaagttaag aataaaaaac caaaggatga aaaagaatga
       61 atatgaaaaa gagtagagaa taactttgaa aggggaccat gatataactg gaaaaaagag
      121 gttcttggaa atgaaaagtt accaaagagt atttataatt cagaaaaaaa agccaacgaa
      181 tatcgttttg atggcgagcc tttttttttt ttttaggaag acactaaagg tacctagcat
      241 catatgggaa ggaaaggaaa tcacttggaa gacatcacaa gcattcattt accaagagaa
      301 aaaatatgca ttttagctaa gatccattga acaaagcact cactcaactc aactgaatga
      361 acgaaagaag aaagaacagt agaaaacact ttgtgacggt gcggaacaca tttacgtagc
      421 tatcatgctg aattctacta tgaaaatctc ccaatctgtc gatggcaaaa cgacccacgt
      481 ggcagagttg ggtcaagtgc cagtttctgg attaagtaac agatacagac atcacacgcc
      541 atagaggaat cccgccgttg cgagagatgg aaaacaatag agccgaaatt gtggaagccc
      601 gatgtctggg tgtacatttt ttttttttct ttctttctct ttcaataatc tttccttttt
      661 ccatttagct tgccggaaaa actttcgggt agcgaaaatc tttctgccgg aaaaattagc
      721 tatttttttc ttccttatta tttttttagt tctgaagttt gaccagggcg actagagatc
      781 tcgacactgg atggcggcgt tagtatcgaa tcgacagcag tatagcgacc agcattcaca
      841 tacgattgac gcatgatatt actttctgcg cacttaactt cgcatctggg cagatgatgt
      901 cgaggcgaaa aaaaatataa atcacgctaa catttgatta aaatagaaca actacaatat
      961 aaaaaaacta tacaaatgac aagttcttga aaacaagaat ctttttattg tcagtactga
     1021 ttattccttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc
     1081 tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt
     1141 cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa
     1201 attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg
     1261 gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc
     1321 catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc
     1381 gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt
     1441 gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag
     1501 catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg
     1561 ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg
     1621 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc
     1681 gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag
     1741 gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa
     1801 ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac
     1861 ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc
     1921 tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga
     1981 gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt
     2041 tttacccatg gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta
     2101 aaaggaagta tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac
     2161 aggggcgcgg cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag
     2221 gtccgcagcg aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat
     2281 gcaaatgatt atacatgggg atgtatgggc taaatgtacg ggcgacagtc acatcatgcc
     2341 cctgagctgc gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctggcc
     2401 gggtgacccg gcggggacgt cgagaaccct taatataact tcgtataatg tatgctatac
     2461 gaagttatta ggtgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct
     2521 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     2581 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2641 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2701 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2761 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2821 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2881 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2941 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3001 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3061 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3121 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3181 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3241 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3301 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3361 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3421 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3481 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3541 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3601 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3661 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3721 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3781 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3841 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3901 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3961 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4021 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4081 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4141 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4201 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4261 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4321 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4381 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4441 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4501 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4561 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     4621 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4681 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4741 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4801 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     4861 acatacgatt taggtgacac tatagaacgc ggccgccagg atctcgcggc cgcccatgga
     4921 tcctttatcg tcgtcgtcct tgtaatcctt atcgtcgtcg tccttataat ctttgtcgtc
     4981 gtcgtcttta tagtccatgg tgtcgacaac ccttaatata acttcgtata atgtatgcta
     5041 tacgaagtta ttaggtctag atgtgatgat gttttatttg ttttgattgg tgtcttgtaa
     5101 ata
//
