LOCUS       sequence-203254-        5100 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 5100)
  AUTHORS   Wang N, Lo Presti L, Zhu YH, Kang M, Wu Z, Martin SG, Wu JQ
  TITLE     The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during 
            fission yeast cytokinesis.
  JOURNAL   J Cell Biol. 2014 May 12;205(3):357-75. doi: 10.1083/jcb.201308146. 
            Epub 2014 May 5.
  PUBMED    24798735
REFERENCE   2  (bases 1 to 5100)
  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: 
            "10.1083/jcb.201308146"; journalName: "J Cell Biol"; date: 
            "2014-05-12- 12"; volume: "205"; issue: "3"; pages: "357-75"
COMMENT     Sequence Label: pFA6a-mCherry-hphMX6
FEATURES             Location/Qualifiers
     source          1..5100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..24
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6..23
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             85..795
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(230..248)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     498..517
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      816..1003
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(871..895)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1078..2653
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1078..1421
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1422..2450
                     /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      2456..2653
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2759..2778)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2760..2778)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2865..2882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3036..3624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3116..3135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3795..4586,4587..4655))
                     /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     4418..4437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4656..4760)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4828..4846
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4884..4906)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5006..5025
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacgattta ggtgacacta tagaacgcgg ccgccagctg aagcttcgta cgctgcaggt
       61 cgacggatcc ccgggttaat taaaatggtg agcaagggcg aggaggataa catggccatc
      121 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
      181 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
      241 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
      301 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
      361 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
      421 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
      481 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
      541 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
      601 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
      661 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
      721 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
      781 gagctgtaca agtaaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt
      841 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
      901 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
      961 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctagggata
     1021 acagggtaat atagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac
     1081 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt
     1141 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat
     1201 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcggac ctgcgagcag
     1261 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat
     1321 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg
     1381 ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaaa aagcctgaac
     1441 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     1501 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     1561 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     1621 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     1681 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     1741 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     1801 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     1861 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     1921 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     1981 aggactgccc cgaagtccgg cacctcatgc acgcggattt cggctccaac aatgtcctga
     2041 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     2101 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     2161 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     2221 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     2281 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     2341 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     2401 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa tcagtactga
     2461 cagtaaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt
     2521 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg
     2581 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct
     2641 ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtgttta aacgagctcg
     2701 aattcatcga tgatatcaga tccactagtg gcctatgcgg ccgcggatct gccggtctcc
     2761 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     2821 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2881 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2941 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3001 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3061 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3241 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3361 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3421 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3481 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3541 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3601 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3661 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3721 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3781 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3841 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3901 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3961 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4021 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4081 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4141 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4201 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4261 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4321 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4381 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4441 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4501 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4561 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4621 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4681 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4741 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4801 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     4861 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4921 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4981 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5041 atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
//
