LOCUS       sequence-204151-        5820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast gene targeting.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5820)
  AUTHORS   Zhu YH, Ye Y, Wu Z, Wu JQ
  TITLE     Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the
            regulation of fission yeast cytokinesis.
  JOURNAL   Mol Biol Cell. 2013 Oct;24(20):3187-204. doi: 
            10.1091/mbc.E13-06-0301. Epub 2013 Aug 21.
  PUBMED    23966468
REFERENCE   2  (bases 1 to 5820)
  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"; doi: 
            "10.1091/mbc.E13-06-0301"; journalName: "Mol Biol Cell"; date: 
            "2013-10"; volume: "24"; issue: "20"; pages: "3187-204"
COMMENT     Sequence Label: pFA6a-tdTomato-hphMX6
FEATURES             Location/Qualifiers
     source          1..5820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..26
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     8..25
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             87..1517
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     terminator      1538..1725
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1593..1617)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1800..3375
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1800..2143
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2144..3172
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLMHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      3178..3375
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3481..3500)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3482..3500)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3587..3604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3758..4346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3838..3857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4517..5308,5309..5377))
                     /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     5140..5159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5378..5482)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5550..5568
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5606..5628)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5728..5747
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acatacgatt taggtgacac tatagaacgc ggccgccagc tgaagcttcg tacgctgcag
       61 gtcgacggat ccccgggtta attaaaatgg tgagcaaggg cgaggaggtc atcaaagagt
      121 tcatgcgctt caaggtgcgc atggagggct ccatgaacgg ccacgagttc gagatcgagg
      181 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
      241 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttcatgtac ggctccaagg
      301 cgtacgtgaa gcaccccgcc gacatccccg attacaagaa gctgtccttc cccgagggct
      361 tcaagtggga gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg acccaggact
      421 cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttccccc
      481 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
      541 acccccgcga cggcgtgctg aagggcgaga tccaccaggc cctgaagctg aaggacggcg
      601 gccactacct ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg
      661 gctactacta cgtggacacc aagctggaca tcacctccca caacgaggac tacaccatcg
      721 tggaacagta cgagcgctcc gagggccgcc accacctgtt cctggggcat ggcaccggca
      781 gcaccggcag cggcagctcc ggcaccgcct cctccgagga caacaacatg gccgtcatca
      841 aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga
      901 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
      961 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct
     1021 ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg
     1081 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc
     1141 aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact
     1201 tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc
     1261 gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg
     1321 acggcggccg ctacctggtg gagttcaaga ccatctacat ggccaagaag cccgtgcaac
     1381 tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca
     1441 ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg tacggcatgg
     1501 acgagctgta caagtaaggc gcgccacttc taaataagcg aatttcttat gatttatgat
     1561 ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa gtgactctta
     1621 ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca ggttgctttc
     1681 tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagat ccgctaggga
     1741 taacagggta atatagatct gtttagcttg cctcgtcccc gccgggtcac ccggccagcg
     1801 acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat
     1861 gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac
     1921 attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcgg acctgcgagc
     1981 agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa
     2041 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     2101 tgctaggata cagttctcac atcacatccg aacataaaca accatgggta aaaagcctga
     2161 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     2221 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     2281 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     2341 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     2401 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     2461 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     2521 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     2581 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     2641 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     2701 cgaggactgc cccgaagtcc ggcacctcat gcacgcggat ttcggctcca acaatgtcct
     2761 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     2821 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     2881 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     2941 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     3001 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     3061 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     3121 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aatcagtact
     3181 gacagtaaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta
     3241 gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc
     3301 tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg
     3361 ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtt taaacgagct
     3421 cgaattcatc gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct
     3481 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     3541 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3601 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3661 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3721 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3781 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3841 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3901 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3961 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4021 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4081 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4141 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4201 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4261 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4321 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4381 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4441 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4501 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4561 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4621 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4681 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4741 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4801 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4861 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4921 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4981 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5041 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5101 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5161 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5221 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5281 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5341 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5401 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5461 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5521 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     5581 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     5641 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     5701 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     5761 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
//
