LOCUS       sequence-146319-        4492 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 4492)
  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 4492)
  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: pNX3a2-PK3
FEATURES             Location/Qualifiers
     source          1..4492
                     /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"
     terminator      564..751
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(619..643)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     polyA_signal    771..852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     796..815
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(871..1665)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(982..1001)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1592..1611
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1720..1767)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(1864..1888)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1885..2088)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1914..1934)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2089..2392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2466..2485)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2467..2485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2572..2589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2743..3331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2823..2842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3502..4293,4294..4362))
                     /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     4125..4144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4363..4467)
                     /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 tacctctaga
      541 tgaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
      601 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
      661 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg
      721 aggtcgctct tattgaccac acctctaccg gcagatccgc taggagatct gcagtgaaaa
      781 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
      841 aataaacaag ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
      901 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
      961 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     1021 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     1081 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc
     1141 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     1201 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     1261 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     1321 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     1381 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     1441 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
     1501 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     1561 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     1621 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatccagc
     1681 tttttgcaaa agcctagcca tggtttagtt cctcaccttg tcgtattata ctatgccgat
     1741 atactatgcc gatgattaat tgtcaacacg gtccgttcca atgcaccgtt cccggccgcg
     1801 gaggctggat cggtcccggt gtcttctatg gaggtcaaaa cagcgtggat ggcgtctcca
     1861 ggcgatctga cggttcacta aacgagctct gcttatatag acctcccacc gtacacgcct
     1921 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
     1981 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     2041 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
     2101 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     2161 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     2221 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     2281 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     2341 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cggtttaaac
     2401 gagctcgaat tcatcgatga tatcagatcc actagtggcc tatgcggccg cggatctgcc
     2461 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     2521 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2581 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2641 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2701 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2761 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2821 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2881 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     2941 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3001 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3061 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3121 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3181 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3241 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3301 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3361 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3421 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3481 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3541 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3601 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3661 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3721 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3781 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3841 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3901 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3961 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4021 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4081 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4141 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4201 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4261 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4321 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4381 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4441 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ct
//
