LOCUS       sequence-271032-        3996 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 3996)
  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 3996)
  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:natMX4
FEATURES             Location/Qualifiers
     source          1..3996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..112
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        193..211
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     193..210
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             307..330
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      354..541
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(409..433)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            616..1735
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        616..959
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             960..1532
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1538..1735
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1842..1861)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1843..1861)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1948..1965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2119..2707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2199..2218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2878..3669,3670..3738))
                     /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     3501..3520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3739..3843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3911..3929
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3967..3989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
       61 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
      121 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
      181 catacacata cgatttaggt gacactatag aacgcggccg ccagctgaag cttcgtacgc
      241 tgcaggtcga cggatccccg ggttaattaa ggggatagcc tggacatgct cgagtggtct
      301 ctgatggatt acaaggacga cgatgacaag taaggcgcgc cacttctaaa taagcgaatt
      361 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
      421 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
      481 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
      541 gcagatccgc tagggataac agggtaatat agatctgttt agcttgcctc gtccccgccg
      601 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
      661 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
      721 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
      781 ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
      841 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
      901 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
      961 tgggtaccac tcttgacgac acggcttacc ggtaccgcac cagtgtcccg ggggacgccg
     1021 aggccatcga ggcactggat gggtccttca ccaccgacac cgtcttccgc gtcaccgcca
     1081 ccggggacgg cttcaccctg cgggaggtgc cggtggaccc gcccctgacc aaggtgttcc
     1141 ccgacgacga atcggacgac gaatcggacg acggggagga cggcgacccg gactcccgga
     1201 cgttcgtcgc gtacggggac gacggcgacc tggcgggctt cgtggtcgtc tcgtactccg
     1261 gctggaaccg ccggctgacc gtcgaggaca tcgaggtcgc cccggagcac cgggggcacg
     1321 gggtcgggcg cgcgttgatg gggctcgcga cggagttcgc ccgcgagcgg ggcgccgggc
     1381 acctctggct ggaggtcacc aacgtcaacg caccggcgat ccacgcgtac cggcggatgg
     1441 ggttcaccct ctgcggcctg gacaccgccc tgtacgacgg caccgcctcg gacggcgagc
     1501 aggcgctcta catgagcatg ccctgcccct aatcagtact gacaataaaa agattcttgt
     1561 tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa
     1621 tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     1681 tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     1741 gattcgatac taacgccgcc atccagtgtc gaaaacgagc tcgaattcat cgatgatatc
     1801 agatccacta gtggcctatg cggccgcgga tctgccggtc tccctatagt gagtcgtatt
     1861 aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1921 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2521 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2641 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3181 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3481 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3541 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3601 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3661 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3721 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3781 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3841 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     3901 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     3961 tgaaaacctc tgacacatgc agctcccgga gacggt
//
