LOCUS       sequence-178209-        4834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pH3FS-PYK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4834)
  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 4834)
  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: pH3FS-PYK1
FEATURES             Location/Qualifiers
     source          1..4834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    57..90
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(978..2006)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(978..2006)
                     /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(2007..2350)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2392..2425
                     /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(2482..2501)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2483..2501)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2588..2605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2759..3347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2839..2858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3518..4309,4310..4378))
                     /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     4141..4160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4379..4483)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4551..4569
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4607..4629)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4729..4748
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(4797..4834,1..34))
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
ORIGIN
        1 ccttataatc tttgtcgtcg tcgtctttat agtccatggt gtcgacaacc cttaatataa
       61 cttcgtataa tgtatgctat acgaagttat taggtctaga tgtgttgatg ttttatttgt
      121 tttgattggt gtcttgtaaa tagaaacaag agagaataat aaacaagtta agaataaaaa
      181 accaaaggat gaaaaagaat gaatatgaaa aagagtagag aataactttg aaaggggacc
      241 atgatataac tggaaaaaag aggttcttgg aaatgaaaag ttaccaaaga gtatttataa
      301 ttcagaaaaa aaagccaacg aatatcgttt tgatggcgag cctttttttt tttttaggaa
      361 gacactaaag gtacctagca tcatatggga aggaaaggaa atcacttgga agacatcaca
      421 agcattcatt taccaagaga aaaaatatgc attttagcta agatccattg aacaaagcac
      481 tcactcaact caactgaatg aacgaaagaa gaaagaacag tagaaaacac tttgtgacgg
      541 tgcggaacac atttacgtag ctatcatgct gaattctact atgaaaatct cccaatctgt
      601 cgatggcaaa acgacccacg tggcagagtt gggtcaagtg ccagtttctg gattaagtaa
      661 cagatacaga catcacacgc catagaggaa tcccgccgtt gcgagagatg gaaaacaata
      721 gagcagaaat tgtggaagcc cgatgtctgg gtgtacattt tttttttctt tctttctctt
      781 tcaataatct ttcctttttc catttagctt gccggaaaaa ctttcgggta gcgaaaatct
      841 ttctgccgga aaaattagct atttttttct tccttattat ttttttagtt ctgaagtttg
      901 accagggcga ctagagatct aaaagtcagg cgatcacaca ttaaaagcta tacattacaa
      961 agcgttaata gcgggcatta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca
     1021 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     1081 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     1141 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     1201 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     1261 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     1321 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     1381 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     1441 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     1501 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     1561 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     1621 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     1681 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     1741 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     1801 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     1861 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     1921 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     1981 gcggtgagtt caggcttttt acccatggtt gtttatgttc ggatgtgatg tgagaactgt
     2041 atcctagcaa gattttaaaa ggaagtatat gaaagaagaa cctcagtggc aaatcctaac
     2101 cttttatatt tctctacagg ggcgcggcgt ggggacaatt caacgcgtct gtgaggggag
     2161 cgtttccctg ctcgcaggtc cgcagcgagg agccgtaatt tttgcttcgc gccgtgcggc
     2221 catcaaaatg tatggatgca aatgattata catggggatg tatgggctaa atgtacgggc
     2281 gacagtcaca tcatgcccct gagctgcgca cgtcaagact gtcaaggagg gtattctggg
     2341 cctccatgtc gctggccggg tgacccggcg gggacgtcga gaacccttaa tataacttcg
     2401 tataatgtat gctatacgaa gttattaggt gatatcagat ccgtcgacct gcagcgtacg
     2461 cggccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta
     2521 acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2581 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2641 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2701 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2761 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2821 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2881 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2941 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3001 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3061 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3121 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3181 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3241 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3301 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3361 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3421 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3481 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3541 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3601 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3661 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3721 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3781 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3841 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3901 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3961 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4021 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4081 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4141 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4201 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4261 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4321 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4381 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4441 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4501 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     4561 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     4621 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     4681 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     4741 agattgtact gagagtgcac ctcgagcatc gatgaattct ctgtcgccat ggatccttta
     4801 tcgtcgtcgt ccttgtaatc cttatcgtcg tcgt
//
