LOCUS       sequence-169917-        4066 bp    DNA     circular SYN 14-APR-2025
DEFINITION  N-ICE plasmid pK3F.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4066)
  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 4066)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..4066
                     /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)."
     gene            complement(142..1498)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(142..339)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(345..1154)
                     /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     1068..1087
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1155..1498)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    1540..1573
                     /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(1626..1645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1627..1645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1732..1749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1903..2491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1983..2002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2662..3453,3454..3522))
                     /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     3285..3304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3523..3627)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3695..3713
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3751..3773)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3873..3892
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3973..3991
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3973..3990
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(4029..4066,1..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 gatctcgaca ctggatggcg
      121 gcgttagtat cgaatcgaca gcagtatagc gaccagcatt cacatacgat tgacgcatga
      181 tattactttc tgcgcactta acttcgcatc tgggcagatg atgtcgaggc gaaaaaaaat
      241 ataaatcacg ctaacatttg attaaaatag aacaactaca atataaaaaa actatacaaa
      301 tgacaagttc ttgaaaacaa gaatcttttt attgtcagta ctgattagaa aaactcatcg
      361 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
      421 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
      481 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
      541 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
      601 ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
      661 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
      721 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
      781 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
      841 aatgctgttt tgccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
      901 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
      961 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     1021 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     1081 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga aacgtgagtc
     1141 ttttccttac ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga
     1201 ttttaaaagg aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc
     1261 tctacagggg cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct
     1321 cgcaggtctg cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta
     1381 tggatgcaaa tgattataca tggggatgta tgggctaaat gtacgggcga cagtcacatc
     1441 atgcccctga gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgc
     1501 tggccgggtg acccggcggg gacgtcgaga acccttaata taacttcgta taatgtatgc
     1561 tatacgaagt tattaggtga tatcagatcc actagtggcc tatgcggccg cggatctgcc
     1621 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     1681 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1741 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1801 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1861 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1921 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1981 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2041 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2101 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2161 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2221 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2281 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2341 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2401 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2461 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2521 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2581 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2641 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2701 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2761 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2821 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2881 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2941 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3001 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3061 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3121 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3181 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3241 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3301 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3361 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3421 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3481 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3541 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3601 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     3661 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3721 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3781 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3841 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     3901 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     3961 catacacata cgatttaggt gacactatag aacgcggccg ccaggatctc gcggccgccc
     4021 atggatcctt tatcgtcgtc gtccttgtaa tccttatcgt cgtcgt
//
