LOCUS       sequence-153387-        4004 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 4004)
  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 4004)
  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: pNX3c-FL3
FEATURES             Location/Qualifiers
     source          1..4004
                     /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      519..706
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(574..598)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            781..1900
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        781..1124
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1125..1697
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1703..1900
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2005..2024)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2006..2024)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2084..2101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2255..2843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2335..2354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3014..3805,3806..3874))
                     /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     3637..3656
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3875..3979)
                     /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 gagattgagg cgcgccactt ctaaataagc gaatttctta tgatttatga
      541 tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt
      601 aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt
      661 ctcaggtata gtatgaggtc gctcttattg accacacctc taccggcaga tccgctaggg
      721 ataacagggt aatatagatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc
      781 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
      841 tgtgactgtc gcccgtatat ttagcccaca catccccatg tataatcatt tgcatccata
      901 cattttgatg gctgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag
      961 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
     1021 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
     1081 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt accactcttg
     1141 acgacacggc ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac
     1201 tggatgggtc cttcaccacc gacaccgtct tccgcgtcac cgccaccggg gacggcttca
     1261 ccctgcggga ggtgccggtg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg
     1321 acgacgaatc ggacgacggg gaggacggcg acccggactc ccggacgttc gtcgcgtacg
     1381 gggacgacgg cgacctggcg ggcttcgtgg tcgtctcgta ctccggctgg aaccgccggc
     1441 tgaccgtcga ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt
     1501 tgatggggct cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg
     1561 tcaccaacgt caacgcaccg gcgatccacg cgtaccggcg gatggggttc accctctgcg
     1621 gcctggacac cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga
     1681 gcatgccctg cccctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc
     1741 atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta
     1801 tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat
     1861 atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg
     1921 ccgccatcca gtgttaaacg agctcgaatt catcgatgat atcagatcca ctagtggcct
     1981 atgcggccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2041 ttaacctgca ttaatgaatc ggccaacgcg cggggtaggc ttcctcgctc actgactcgc
     2101 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2161 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2221 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2281 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2341 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2401 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     2461 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2521 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2581 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2641 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     2701 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2761 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2821 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2881 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2941 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3001 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3061 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3121 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3181 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3241 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3301 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3361 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3421 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3481 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3541 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3601 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3661 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3721 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3781 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3841 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3901 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3961 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acct
//
