LOCUS       sequence-160584-        4499 bp    DNA     circular SYN 15-APR-2025
DEFINITION  fission yeast epitope tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4499)
  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 4499)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pNX3b-PK6
FEATURES             Location/Qualifiers
     source          1..4499
                     /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             406..447
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             451..492
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             493..534
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             541..582
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             586..627
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             628..669
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     terminator      699..886
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(754..778)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            945..2520
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        945..1288
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1289..2317
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLMHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2323..2520
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2579..2596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2750..3338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2830..2849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3509..4300,4301..4369))
                     /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     4132..4151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4370..4474)
                     /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 ctagcggtaa gcctattcct
      421 aaccctcttt tgggtctcga ttctacaggt ggaaaaccaa tccccaaccc actcctcggc
      481 cttgactcaa ctggcaaacc cataccaaat cctttacttg gattagatag tacctctagc
      541 ggtaagccta ttcctaaccc tcttttgggt ctcgattcta caggtggaaa accaatcccc
      601 aacccactcc tcggccttga ctcaactggc aaacccatac caaatccttt acttggatta
      661 gatagtacct ctagatgagg cgcgccactt ctaaataagc gaatttctta tgatttatga
      721 tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt
      781 aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt
      841 ctcaggtata gtatgaggtc gctcttattg accacacctc taccggcaga tccgctagga
      901 gatctgttta gcttgcctcg tccccgccgg gtcacccggt cagcgacatg gaggcccaga
      961 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
     1021 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
     1081 cggcgcgaag caaaaattac ggctcctcgc tgcggacctg cgagcaggga aacgctcccc
     1141 tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg
     1201 atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt
     1261 ctcacatcac atccgaacat aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc
     1321 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     1381 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     1441 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     1501 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     1561 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     1621 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1681 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1741 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     1801 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     1861 agtccggcac ctcatgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     1921 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     1981 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     2041 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     2101 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     2161 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     2221 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     2281 ccgacgcccc agcactcgtc cgagggcaaa ggaataatca gtactgacaa taaaaagatt
     2341 cttgttttca agaacttgtc atttgtatag tttttttaca ttgtagttgt tctattttaa
     2401 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     2461 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     2521 ctgtcgattc gatactaacg ccgccatcca gtttaaacga gctcgaattc taggcttcct
     2581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2941 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3181 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacct
//
