LOCUS       sequence-203263-        5275 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 5275)
  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 5275)
  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-kanMX6
FEATURES             Location/Qualifiers
     source          1..5275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     21..38
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             109..450
                     /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             109..450
                     /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             517..1191
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             517..1191
                     /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(647..665)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     915..934
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1109..1132
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      1227..1414
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1282..1306)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1489..2845
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1489..1832
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1833..2642
                     /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     complement(1900..1919)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2648..2845
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2949..2968)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2950..2968)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3055..3072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3226..3814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3306..3325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3985..4776,4777..4845))
                     /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     4608..4627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4846..4950)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5018..5036
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5074..5096)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5196..5215
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
       61 tcgtacgctg caggtcgacg gatccccggg ttaattaaca tggccgatgt gcagctggtg
      121 gagtctgggg gagccttggt gcagccgggg gggtctctga gactctcctg tgcagcctct
      181 ggattccccg tcaatcgcta tagtatgagg tggtaccgcc aggctccagg gaaggagcgc
      241 gagtgggtcg cgggtatgag tagtgctggt gatcgttcaa gttatgaaga ctccgtgaag
      301 ggccgattca ccatctccag agacgacgcc aggaatacgg tgtatctgca aatgaacagc
      361 ctgaaacctg aggacacggc cgtgtattac tgtaatgtca atgtgggctt tgagtactgg
      421 ggccagggga cccaggtcac cgtctcctca aggaattcga tatcaagctt atcgataccg
      481 tcgacggtac cgcgggcccg ggatccaccg gtcgacatgg cctcctccga ggacgtcatc
      541 aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca cgagttcgag
      601 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
      661 accaagggcg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt ccagtacggc
      721 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
      781 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
      841 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
      901 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctccaccgag
      961 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agatgaggct gaagctgaag
     1021 gacggcggcc actacgacgc cgaggtcaag accacctaca tggccaagaa gcccgtgcag
     1081 ctgcccggcg cctacaagac cgacatcaag ctggacatca cctcccacaa cgaggactac
     1141 accatcgtgg aacagtacga gcgcgccgag ggccgccact ccaccggcgc cggcctgtac
     1201 aagtaaggcg cgccacttct aaataagcga atttcttatg atttatgatt tttattatta
     1261 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     1321 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt
     1381 atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa
     1441 tatagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc
     1501 cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc
     1561 ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc
     1621 cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga cctgcgagca gggaaacgct
     1681 cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt
     1741 taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac
     1801 agttctcaca tcacatccga acataaacaa ccatgggtaa ggaaaagact cacgtttcga
     1861 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     1921 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     1981 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     2041 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     2101 atgatgcatg gttactcacc actgcgatcc ccggcaaaac agcattccag gtattagaag
     2161 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     2221 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     2281 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     2341 gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc tcaccggatt
     2401 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     2461 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     2521 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     2581 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     2641 aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt
     2701 ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct
     2761 cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg
     2821 tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagttta
     2881 aacgagctcg aattcatcga tgatatcaga tccactagtg gcctatgcgg ccgcggatct
     2941 gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg
     3001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3661 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4861 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4921 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4981 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     5041 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     5101 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     5161 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     5221 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taacc
//
