LOCUS       sequence-271040-        4452 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 4452)
  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 4452)
  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:hphMX4
FEATURES             Location/Qualifiers
     source          1..4452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..86
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(124..146)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     246..265
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        346..364
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     346..363
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             460..483
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      507..694
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(562..586)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            769..2344
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        769..1112
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1113..2141
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1113..2141
                     /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      2147..2344
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2451..2470)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2452..2470)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2557..2574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2728..3316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2808..2827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3487..4278,4279..4347))
                     /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     4110..4129
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4348..4452)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
       61 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
      121 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
      181 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
      241 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
      301 acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
      361 tagaacgcgg ccgccagctg aagcttcgta cgctgcaggt cgacggatcc ccgggttaat
      421 taaggggata gcctggacat gctcgagtgg tctctgatgg attacaagga cgacgatgac
      481 aagtaaggcg cgccacttct aaataagcga atttcttatg atttatgatt tttattatta
      541 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
      601 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt
      661 atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa
      721 tatagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc
      781 cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc
      841 ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc
      901 cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca gggaaacgct
      961 cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt
     1021 taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac
     1081 agttctcaca tcacatccga acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga
     1141 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     1201 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     1261 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     1321 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     1381 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     1441 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     1501 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     1561 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     1621 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     1681 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     1741 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     1801 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     1861 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     1921 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     1981 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     2041 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     2101 gaaaccgacg ccccagcact cgtccgaggg caaaggaata atcagtactg acaataaaaa
     2161 gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt
     2221 ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc
     2281 gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat
     2341 actgctgtcg attcgatact aacgccgcca tccagtgtcg aaaacgagct cgaattcatc
     2401 gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg
     2461 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
     2521 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2581 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2641 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2701 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2761 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2821 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2881 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2941 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3001 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3061 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3121 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3181 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3241 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3301 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3361 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3421 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3481 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3541 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3601 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3661 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3721 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3781 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3841 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3901 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3961 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4021 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4081 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4141 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4201 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4261 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4321 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4381 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4441 aggggttccg cg
//
