LOCUS       sequence-9005-ex        5623 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 5623)
  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 5623)
  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-hphMX6-P81nmt1-3FLAG
FEATURES             Location/Qualifiers
     source          1..5623
                     /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..4045)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(2470..2667)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2673..3701)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(2673..3701)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3702..4045)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        4090..5248
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     CDS             5266..5337
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     terminator      5361..5548
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5416..5440)
                     /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 gattattcct ttgccctcgg acgagtgctg
     2701 gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg
     2761 cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg
     2821 catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt
     2881 tggtcaagac caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga
     2941 tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag
     3001 aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc
     3061 gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg
     3121 tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg
     3181 gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc
     3241 gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg
     3301 aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatggcctc cgcgaccggc
     3361 tgcagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg
     3421 cgggagatgc aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc
     3481 gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg
     3541 cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat
     3601 tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac
     3661 ttctcgacag acgtcgcggt gagttcaggc tttttaccca tggttgttta tgttcggatg
     3721 tgatgtgaga actgtatcct agcaagattt taaaaggaag tatatgaaag aagaacctca
     3781 gtggcaaatc ctaacctttt atatttctct acaggggcgc ggcgtgggga caattcaacg
     3841 cgtctgtgag gggagcgttt ccctgctcgc aggtctgcag cgaggagccg taatttttgc
     3901 ttcgcgccgt gcggccatca aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg
     3961 gctaaatgta cgggcgacag tcacatcatg cccctgagct gcgcacgtca agactgtcaa
     4021 ggagggtatt ctgggcctcc atgtcgctgg ccgggtgacc cggcggggac gaggcaagct
     4081 aaacagatct cgccataaaa gacagaataa gtcatcagcg gttgtttcat ttcctatatt
     4141 ttttttttat ttttttattt tttaataagg gaaaatttaa cgtctaagga tacagaagat
     4201 tgttagcaca ttaaagtaat aaaggcttaa gtagtaagtg ccttagcatg ttattgtatt
     4261 tcaaaggaca taatctaaaa taataacaat atcatttctc acaagttatt caattttctt
     4321 ttttttttct aataatatca agaatgtatt atttgtttga cataagtcaa ctaatttatt
     4381 taatatgctg gattaatctt gcagacatgt aaattaacaa gttttagtca aataacgttg
     4441 aagtttcaat gaactcaaat aatttctctt tttttttata taaccatata tgtctaatct
     4501 gatttatatt ttccgcagga tcaactgaag ttatgacatt tggattggat cacttataac
     4561 cttggtcgcc aaataataca aaaatcagcg ttataaaaca aagaaggttt ttgttaagaa
     4621 attaatcctc tttcttgata agaaagttga accgaaattg cagatactga tatatgaaaa
     4681 taatacccac aattttggga atagcgcaag cctcaattta aacaataggt gaggacacat
     4741 gataatgacc tcaatgattg ttagaagaaa agagcctcat tacaaaatcg aaaaatgaat
     4801 ggttgggtac aagtttccaa aacatggtaa agtggacttt gcgtatgaga cgtaaataga
     4861 aaaaaacact tgttatatgt tttctagaat tattgttgtc tctttatggt tggatgatgc
     4921 aaaatagtaa tttcggttag ttgctgtaaa acaccacgag acaaatagat atggatattt
     4981 attaaatcag gaaaaacgta actctcggct actggatggt tcagtcaccc aacgattact
     5041 ggggagagaa aacagggcaa aagcaaagct taaaggaatc cgattgtcat tcggcaatgt
     5101 gcagcgaaac taaaaaccgg ataatggacc tgttaatcga aacattgaag atggaagagg
     5161 aatcctggca tatcatcaat tgaataagtt gaattaatta tttcaatctc attctcactt
     5221 tctgacttat agtcgctttg ttaaatcatg tctttaatta atatggacta caaggacgac
     5281 gatgacaagg attacaaaga tgacgacgat aaggactata aggacgatga tgataaatag
     5341 ggcgcgccac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
     5401 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     5461 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     5521 tcgctcttat tgaccacacc tctaccggca gatccgctag ggataacagg gtaatataga
     5581 tccgtcgacc tgcagcgtac gaagcttcag ctggcggccg cgt
//
