LOCUS       sequence-146302-        4447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fission yeast epitope tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4447)
  AUTHORS   Amelina H, Moiseeva V, Collopy LC, Pearson SR, Armstrong CA, Tomita 
            K
  TITLE     Sequential and counter-selectable cassettes for fission yeast.
  JOURNAL   BMC Biotechnol. 2016 Nov 8;16(1):76.
  PUBMED    27825338
REFERENCE   2  (bases 1 to 4447)
  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: "BMC 
            Biotechnol."; date: "2016-11-8"; volume: "16(1)"; pages: "76"
COMMENT     Sequence Label: pNX3d-FL3
FEATURES             Location/Qualifiers
     source          1..4447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(99..121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        321..339
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     321..338
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             412..435
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             439..486
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      516..703
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(571..595)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             1268..2062
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     primer_bind     complement(2527..2544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2698..3286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2778..2797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3457..4248,4249..4317))
                     /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     4080..4099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4318..4422)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      121 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      181 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
      301 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
      361 tcgtacgctg caggtcgacg gatccccggg ttaattaacg ctagcatggg tgactacaag
      421 gacgacgatg acaagggaga ttacaaagat gacgacgata aggactacaa ggacgacgat
      481 gacaagtcta gatgaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt
      541 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
      601 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
      661 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctaggagat
      721 ctgagctcga tatcaagctt agctacaaat cccactggct atatgtatgc atttgtgtta
      781 aaaaagtttg tatagattat ttaatctact cagcattctt tctctaaata ggaatttgtt
      841 acttaatgga gaaaaaaatg tttcgattta cctagtgtat ttgtttgtat actcacgttt
      901 aatttcaaac atccattcta tcttgtgtaa tttttggcat ggtgaaaaag ataatcagcc
      961 ttataatctt tacaaaagta agaaattctg taaataagcc ttaatgccct tgctttaaat
     1021 taaaatggtt ctttttcatg ataatgtttg cactttgtga atatatttta gatagttctg
     1081 tgaggtataa ttaagatgtt ttagagactt atacaatttt gtctttataa attcttaatt
     1141 gattttacca tcccagttta actatgcttc gtcggcatct ctgcacatgt cgtgttttct
     1201 taccgtattg tcctaccaag aacctctttt ttgcttggat cgaaattaaa ggtttaaaag
     1261 caaagttatg gatgctagag tatttcaaag ctattcagct agagctgagg ggatgaaaaa
     1321 tcccattgcc aaggaattgt tggctttgat ggaagaaaag caaagcaact tgtcagtcgc
     1381 ggtcgatttg acgaagaaat ccgaaatctt agaattggta gataaaattg gaccctatgt
     1441 ctgtgttatc aagacacata ttgacgttgt cgaggatttc gaccaggata tggtagaaaa
     1501 actggtggcc ttaggtaaaa agcatcgttt tcttatcttt gaggatcgca aattcgcaga
     1561 cattggaaat accgtcaagc tacaatatgc atctggtgtg tacaaaattg cttcttgggc
     1621 tcatatcaca aattgccata cagtgccagg cgagggtatt atacaaggcc tcaaagaagt
     1681 tggtttacct ttgggacgtg gtctcttgct tttggctgaa atgtcttcca aaggctcttt
     1741 ggctactggt tcctacacag agaaaacctt agaatggttt gagaagcata ccgatttttg
     1801 ctttggcttt atagctggtc gtcgatttcc taaccttcaa agcgactaca taactatgtc
     1861 ccctggtatc ggcttggatg ttaaaggaga cgggctggga cagcaatatc gtactcctga
     1921 agaagtgatt gtaaactgcg gtagcgatat catcattgtt ggtcgtggag tctatggagc
     1981 tggtcgtaat cctgttgtcg aagccaagag atatagagaa gctggttgga aggcatatca
     2041 gcaaagactt tctcagcatt aaaaaaagac taatgtaaaa tttttttggt tggttattga
     2101 aaaagtcgat gccttgtttg cgtttgtttt cctaggcgtt ttatgtcaga aggcatttag
     2161 aattagtata caagtactct ttggtaaaat tttatgtagc gactaaaata ttaactatta
     2221 tagataaaca ccttgggaat aaaaagtaat ttgctatagt aatttattaa acatgctcct
     2281 acaacattac cacaatcttt tctcttggat tgacattgaa taagaaaaga gtgaattttt
     2341 ttagacttgt aatgataact atgtacaaag ccaatgaaag atgtatgtag atgaatgtaa
     2401 aataccatgt agacaaacaa gataaaactt ggttataaac attggtgttg gaacagaata
     2461 aattagatgt caaaaagttt cgtcaatatc acaagcttgt ttaaacgagc tcgaattcta
     2521 ggcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2581 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2641 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2701 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2761 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2821 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2881 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2941 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3001 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3061 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3121 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3181 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3241 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3301 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3361 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3421 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3481 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3541 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3601 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3661 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3721 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3781 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3841 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3901 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3961 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4021 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4081 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4141 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4201 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4261 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4321 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4381 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4441 gccacct
//
