LOCUS       sequence-146324-        4444 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 4444)
  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 4444)
  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-FL3
FEATURES             Location/Qualifiers
     source          1..4444
                     /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(823..1617)
                     /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(934..953)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1544..1563
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1672..1719)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(1816..1840)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1837..2040)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1866..1886)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2041..2344
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2418..2437)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2419..2437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2524..2541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2695..3283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2775..2794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3454..4245,4246..4314))
                     /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     4077..4096
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4315..4419)
                     /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 agttcgaacc ccagagtccc gctcagaaga actcgtcaag
      841 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
      901 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
      961 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     1021 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     1081 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     1141 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     1201 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     1261 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     1321 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     1381 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     1441 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     1501 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     1561 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     1621 aaacgatcca gctttttgca aaagcctagc catggtttag ttcctcacct tgtcgtatta
     1681 tactatgccg atatactatg ccgatgatta attgtcaaca cggtccgttc caatgcaccg
     1741 ttcccggccg cggaggctgg atcggtcccg gtgtcttcta tggaggtcaa aacagcgtgg
     1801 atggcgtctc caggcgatct gacggttcac taaacgagct ctgcttatat agacctccca
     1861 ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa
     1921 gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg
     1981 aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac
     2041 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
     2101 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
     2161 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
     2221 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
     2281 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     2341 aacggtttaa acgagctcga attcatcgat gatatcagat ccactagtgg cctatgcggc
     2401 cgcggatctg ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct
     2461 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2521 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2581 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2641 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2701 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2761 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2821 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2881 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2941 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3001 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3061 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3121 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3181 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3241 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3301 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3361 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3421 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3481 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3541 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3601 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3661 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3721 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3781 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3841 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3901 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3961 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4021 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4081 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4141 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4201 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4261 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4321 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4381 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4441 acct
//
