LOCUS       sequence-8991-ex        5411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5411)
  AUTHORS   Noguchi C, Garabedian MV, Malik M, Noguchi E
  TITLE     A vector system for genomic FLAG epitope-tagging in 
            Schizosaccharomyces pombe.
  JOURNAL   Biotechnol J. 2008 Oct;3(9-10):1280-5. doi: 10.1002/biot.200800140.
  PUBMED    18729046
REFERENCE   2  (bases 1 to 5411)
  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: "Biotechnol 
            J."; date: "2008-10"; volume: "3(9-10):1280-5. doi"; pages: " 
            10.1002/biot.200800140"
COMMENT     Sequence Label: pFA6a-kanMX6-P3nmt1-3FLAG
FEATURES             Location/Qualifiers
     source          1..5411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..19)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2..19)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(100..119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     219..241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(279..297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        365..469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(470..538,539..1330)
                     /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     complement(688..707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1501..2089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1990..2009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2243..2260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2347..2366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2347..2365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(2470..3826)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(2470..2667)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2673..3482)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3396..3415
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3483..3826)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        3871..5036
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     CDS             5054..5125
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     terminator      5149..5336
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5204..5228)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 tctatagtgt cacctaaatc gtatgtgtat gatacataag gttatgtatt aattgtagcc
       61 gcgttctaac gacaatatgt ccatatggtg cactctcagt acaatctgct ctgatgccgc
      121 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      181 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      241 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
      301 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
      361 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
      421 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
      481 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
      541 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
      601 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
      661 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
      721 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
      781 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
      841 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
      901 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
      961 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1021 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1081 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1141 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     1201 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     1261 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     1321 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     1381 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     1441 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1501 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1561 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1621 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     1681 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1741 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1801 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1861 ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg
     1921 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1981 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2041 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2101 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2161 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2221 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     2281 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttaa cctggcttat
     2341 cgaaattaat acgactcact atagggagac cggcagatcc gcggccgcat aggccactag
     2401 tggatctgat atcatcgatg aattcgagct cgtttaaact ggatggcggc gttagtatcg
     2461 aatcgacagc agtatagcga ccagcattca catacgattg acgcatgata ttactttctg
     2521 cgcacttaac ttcgcatctg ggcagatgat gtcgaggcga aaaaaaatat aaatcacgct
     2581 aacatttgat taaaatagaa caactacaat ataaaaaaac tatacaaatg acaagttctt
     2641 gaaaacaaga atctttttat tgtcagtact gattagaaaa actcatcgag catcaaatga
     2701 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     2761 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     2821 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     2881 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta
     2941 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     3001 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     3061 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     3121 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttg
     3181 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     3241 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     3301 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     3361 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     3421 aaatcagcat ccatgttgga atttaatcgc ggcctcgaaa cgtgagtctt ttccttaccc
     3481 atggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa
     3541 gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg
     3601 cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgca
     3661 gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg
     3721 attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc
     3781 tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg gccgggtgac
     3841 ccggcgggga cgaggcaagc taaacagatc tcgccataaa agacagaata agtcatcagc
     3901 ggttgtttca tttcctatat ttttttttta tttttttatt ttttaataag ggaaaattta
     3961 acgtctaagg atacagaaga ttgttagcac attaaagtaa taaaggctta agtagtaagt
     4021 gccttagcat gttattgtat ttcaaaggac ataatctaaa ataataacaa tatcatttct
     4081 cacaagttat tcaattttct tttttttttc taataatatc aagaatgtat tatttgtttg
     4141 acataagtca actaatttat ttaatatgct ggattaatct tgcagacatg taaattaaca
     4201 agttttagtc aaataacgtt gaagtttcaa tgaactcaaa taatttctct ttttttttat
     4261 ataaccatat atgtctaatc tgatttatat tttccgcagg atcaactgaa gttatgacat
     4321 ttggattgga tcacttataa ccttggtcgc caaataatac aaaaatcagc gttataaaac
     4381 aaagaaggtt tttgttaaga aattaatcct ctttcttgat aagaaagttg aaccgaaatt
     4441 gcagatactg atatatgaaa ataataccca caattttggg aatagcgcaa gcctcaattt
     4501 aaacaatagg tgaggacaca tgataatgac ctcaatgatt gttagaagaa aagagcctca
     4561 ttacaaaatc gaaaaatgaa tggttgggta caagtttcca aaacatggta aagtggactt
     4621 tgcgtatgag acgtaaatag aaaaaaacac ttgttatatg ttttctagaa ttattgttgt
     4681 ctctttatgg ttggatgatg caaaatagta atttcggtta gttgctgtaa aacaccacga
     4741 gacaaataga tatggatatt tattaaatca ggaaaaacgt aactctcggc tactggatgg
     4801 ttcagtcacc caacgattac tggggagaga aaacagggca aaagcaaagc ttaaaggaat
     4861 ccgattgtca ttcggcaatg tgcagcgaaa ctaaaaaccg gataatggac ctgttaatcg
     4921 aaacattgaa gatatataaa ggaagaggaa tcctggcata tcatcaattg aataagttga
     4981 attaattatt tcaatctcat tctcactttc tgacttatag tcgctttgtt aaatcatgtc
     5041 tttaattaat atggactaca aggacgacga tgacaaggat tacaaagatg acgacgataa
     5101 ggactataag gacgatgatg ataaataggg cgcgccactt ctaaataagc gaatttctta
     5161 tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa
     5221 agtgactctt aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc
     5281 aggttgcttt ctcaggtata gtatgaggtc gctcttattg accacacctc taccggcaga
     5341 tccgctaggg ataacagggt aatatagatc cgtcgacctg cagcgtacga agcttcagct
     5401 ggcggccgcg t
//
