LOCUS       sequence-271017-        4230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR-based C-terminal A1-FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4230)
  AUTHORS   Nemec AA, Tomko RJ Jr
  TITLE     A suite of PCR-based peptide tagging plasmids to permit 
            epitope-targeted enzymatic functionalization of yeast proteins.
  JOURNAL   Yeast. 2020 May 13. doi: 10.1002/yea.3471.
  PUBMED    32401365
REFERENCE   2  (bases 1 to 4230)
  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. 2020
            May 13. doi: 10.1002/yea.3471."
COMMENT     Sequence Label: pFA6a-A1-FLAG:kanMX6
FEATURES             Location/Qualifiers
     source          1..4230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        124..142
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     124..141
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             238..261
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      285..472
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(340..364)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            547..1903
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        547..890
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             891..1700
                     /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(958..977)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1706..1903
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2007..2026)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2008..2026)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2113..2130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2284..2872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2364..2383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3043..3834,3835..3903))
                     /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     3666..3685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3904..4008)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4076..4094
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4132..4154)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
       61 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
      121 acgatttagg tgacactata gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg
      181 acggatcccc gggttaatta aggggatagc ctggacatgc tcgagtggtc tctgatggat
      241 tacaaggacg acgatgacaa gtaaggcgcg ccacttctaa ataagcgaat ttcttatgat
      301 ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg
      361 actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg taggtcaggt
      421 tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc ggcagatccg
      481 ctagggataa cagggtaata tagatctgtt tagcttgcct cgtccccgcc gggtcacccg
      541 gccagcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg
      601 gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca
      661 tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc
      721 tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg
      781 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt
      841 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaagg
      901 aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
      961 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     1021 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     1081 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     1141 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag
     1201 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     1261 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     1321 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     1381 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt
     1441 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     1501 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     1561 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     1621 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     1681 tgctcgatga gtttttctaa tcagtactga caataaaaag attcttgttt tcaagaactt
     1741 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat
     1801 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt
     1861 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta
     1921 acgccgccat ccagtttaaa cgagctcgaa ttcatcgatg atatcagatc cactagtggc
     1981 ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca
     2041 ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2101 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2161 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2221 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2281 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2341 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2401 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2461 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2521 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2581 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2641 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2701 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2761 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2821 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2881 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2941 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3001 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3061 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3121 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3181 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3241 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3301 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3361 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3421 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3481 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3541 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3601 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3661 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3721 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3781 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3841 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3901 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3961 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4021 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4081 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4141 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4201 ccgtcagggc gcgtcagcgg gtgttggcgg
//
