LOCUS       sequence-203265-        5496 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 5496)
  AUTHORS   Davidson R, Laporte D, Wu JQ
  TITLE     Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast 
            cytokinesis.
  JOURNAL   Mol Biol Cell. 2015 Feb 1;26(3):453-66. doi: 
            10.1091/mbc.E14-07-1252. Epub 2014 Dec 3.
  PUBMED    25473118
REFERENCE   2  (bases 1 to 5496)
  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.E14-07-1252"; journalName: "Mol Biol Cell"; date: 
            "2015-02-1- 1"; volume: "26"; issue: "3"; pages: "453-66"
COMMENT     Sequence Label: pFA6a-GBP-RFP-hphMX6
FEATURES             Location/Qualifiers
     source          1..5496
                     /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             96..437
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             96..437
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             504..1178
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             504..1178
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(634..652)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     902..921
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1096..1119
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      1214..1401
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1269..1293)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1476..3051
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1476..1819
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1820..2848
                     /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      2854..3051
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3157..3176)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3158..3176)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3263..3280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3434..4022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3514..3533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4193..4984,4985..5053))
                     /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     4816..4835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5054..5158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5226..5244
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5282..5304)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5404..5423
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acatacgatt taggtgacac tatagaacgc ggccgccagc tgaagcttcg tacgctgcag
       61 gtcgacggat ccccgggtta attaacatgg ccgatgtgca gctggtggag tctgggggag
      121 ccttggtgca gccggggggg tctctgagac tctcctgtgc agcctctgga ttccccgtca
      181 atcgctatag tatgaggtgg taccgccagg ctccagggaa ggagcgcgag tgggtcgcgg
      241 gtatgagtag tgctggtgat cgttcaagtt atgaagactc cgtgaagggc cgattcacca
      301 tctccagaga cgacgccagg aatacggtgt atctgcaaat gaacagcctg aaacctgagg
      361 acacggccgt gtattactgt aatgtcaatg tgggctttga gtactggggc caggggaccc
      421 aggtcaccgt ctcctcaagg aattcgatat caagcttatc gataccgtcg acggtaccgc
      481 gggcccggga tccaccggtc gacatggcct cctccgagga cgtcatcaag gagttcatgc
      541 gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
      601 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
      661 ccctgccctt cgcctgggac atcctgtccc ctcagttcca gtacggctcc aaggcctacg
      721 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
      781 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
      841 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
      901 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgg atgtaccccg
      961 aggacggcgc cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact
     1021 acgacgccga ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg cccggcgcct
     1081 acaagaccga catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     1141 agtacgagcg cgccgagggc cgccactcca ccggcgccgg cctgtacaag taaggcgcgc
     1201 cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     1261 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     1321 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg aggtcgctct
     1381 tattgaccac acctctaccg gcagatccgc tagggataac agggtaatat agatctgttt
     1441 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
     1501 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
     1561 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
     1621 gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg aaacgctccc ctcacagacg
     1681 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
     1741 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
     1801 catccgaaca taaacaacca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     1861 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     1921 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     1981 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     2041 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg
     2101 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     2161 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     2221 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     2281 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     2341 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     2401 cctcatgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     2461 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     2521 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     2581 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     2641 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     2701 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     2761 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     2821 cagcactcgt ccgagggcaa aggaataatc agtactgaca ataaaaagat tcttgttttc
     2881 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     2941 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     3001 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     3061 cgatactaac gccgccatcc agtgtttaaa cgagctcgaa ttcatcgatg atatcagatc
     3121 cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt
     3181 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3241 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3301 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3361 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3841 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4081 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4141 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4201 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4261 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4321 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4381 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4441 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4501 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4561 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4621 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4681 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4741 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4801 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4861 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4921 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4981 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5041 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5101 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5161 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5221 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     5281 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     5341 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     5401 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     5461 gcggctacaa ttaatacata accttatgta tcatac
//
