LOCUS       sequence-146373-        3983 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 3983)
  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 3983)
  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: pNX3s2-FL3
FEATURES             Location/Qualifiers
     source          1..3983
                     /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"
     polyA_signal    723..804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     748..767
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(818..1192)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVCVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(1211..1258)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(1355..1379)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1376..1579)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1405..1425)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1580..1883
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(1957..1976)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1958..1976)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2063..2080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2234..2822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2314..2333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2993..3784,3785..3853))
                     /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     3616..3635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3854..3958)
                     /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 ctgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
      781 attataagct gcaataaaca agtttcgagg tcgagtgtca gtcctgctcc tcggccacga
      841 agtgcacgca gttgccggcc gggtcgcgca gggcgaactc ccgcccccac ggctgctcgc
      901 cgatctcggt catggccggc ccggaggcgt cccggaagtt cgtggacacg acctccgacc
      961 actcggcgta cagctcgtcc aggccgcgca cacacaccca ggccagggtg ttgtccggca
     1021 ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc gtcccggacc acaccggcga
     1081 agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc ggtccagaac tcgaccgctc
     1141 cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt caacttggcc atggtttagt
     1201 tcctcacctt gtcgtattat actatgccga tatactatgc cgatgattaa ttgtcaacac
     1261 ggtccgttcc aatgcaccgt tcccggccgc ggaggctgga tcggtcccgg tgtcttctat
     1321 ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact aaacgagctc
     1381 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     1441 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     1501 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     1561 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     1621 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     1681 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     1741 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     1801 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     1861 catttaccgt aagttatgta acggtttaaa cgagctcgaa ttcatcgatg atatcagatc
     1921 cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt
     1981 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2041 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2101 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2161 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2221 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2281 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2341 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2401 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
     2461 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2521 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2581 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2641 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     2701 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2761 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2821 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2881 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2941 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3001 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3061 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3121 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3181 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3241 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3301 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3361 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3421 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3481 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3541 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3601 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3661 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3721 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3781 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3841 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3901 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3961 catttccccg aaaagtgcca cct
//
