LOCUS       sequence-171114-        4744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-ICE plasmid pH3F-GAL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4744)
  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 4744)
  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-GAL1
FEATURES             Location/Qualifiers
     source          1..4744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..82
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     316..333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     420..439
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        420..438
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(492..525)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            567..2142
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        567..910
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             911..1939
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             911..1939
                     /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"
     terminator      1945..2142
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        2194..2635
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2194..2311
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2606..2629
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    complement(2653..2686)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2709..2780
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     promoter        complement(2818..2836)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2819..2836)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2917..2936)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3036..3058
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3096..3114)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3182..3286
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3287..3355,3356..4147)
                     /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(3505..3524)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4318..4744,1..162)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
       61 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      361 ccgcctctcc ccgcgcgttg gccgattcat taatgcaggt taacctggct tatcgaaatt
      421 aatacgactc actataggga gaccggcaga tccgcggccg cataggccac tagtggatct
      481 gatatcacct aataacttcg tatagcatac attatacgaa gttatattaa gggttctcga
      541 cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc cttgacagtc
      601 ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag cccatacatc
      661 cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga agcaaaaatt
      721 acggctcctc gctgcggacc tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt
      781 gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca ctgaggttct
      841 tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc acatccgaac
      901 ataaacaacc atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
      961 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     1021 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     1081 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     1141 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     1201 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     1261 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     1321 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     1381 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     1441 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     1501 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     1561 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     1621 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     1681 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     1741 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     1801 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     1861 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     1921 tccgagggca aaggaataat cagtactgac aataaaaaga ttcttgtttt caagaacttg
     1981 tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt tagcgtgatt
     2041 tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc gcagaaagta
     2101 atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa
     2161 cgccgccatc cagtgtcgag atctctagta gtacggatta gaagccgccg agcgggcgac
     2221 agccctccga cggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa
     2281 acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt
     2341 tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc aaattaacga
     2401 atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta
     2461 attaatcagc gaagcgatga tttttgatct attaacagat atataaatgg aaaagctgca
     2521 taaccacttt aactaatact ttcaacattt tcagtttgta ttacttctta ttcaaatgtc
     2581 ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa
     2641 actctagacc taataacttc gtatagcata cattatacga agttatatta agggttgtcg
     2701 acaccatgga ctataaagac gacgacgaca aagattataa ggacgacgac gataaggatt
     2761 acaaggacga cgacgataaa ggatccatgg gcggccgcga gatcctggcg gccgcgttct
     2821 atagtgtcac ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg
     2881 ttctaacgac aatatgtcca tatggtgcac tctcagtaca atctgctctg atgccgcata
     2941 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3001 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3061 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3121 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3181 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3241 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3301 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3361 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3421 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3481 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3541 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3601 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3661 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3721 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3781 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3841 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3901 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3961 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4021 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4081 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4141 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4201 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4261 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4321 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4381 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4441 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4501 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4561 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4621 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4681 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4741 aaag
//
