LOCUS       sequence-203262-        4743 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 4743)
  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 4743)
  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-hphMX6
FEATURES             Location/Qualifiers
     source          1..4743
                     /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"
     terminator      461..648
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(516..540)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            723..2298
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        723..1066
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1067..2095
                     /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      2101..2298
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2404..2423)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2405..2423)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2510..2527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2681..3269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2761..2780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3440..4231,4232..4300))
                     /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     4063..4082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4301..4405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4473..4491
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4529..4551)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4651..4670
                     /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 ctcctcataa ggcgcgccac ttctaaataa gcgaatttct tatgatttat
      481 gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc
      541 ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct
      601 ttctcaggta tagtatgagg tcgctcttat tgaccacacc tctaccggca gatccgctag
      661 ggataacagg gtaatataga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca
      721 gcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat
      781 gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca
      841 tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cggacctgcg
      901 agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga
      961 gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa
     1021 tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg gtaaaaagcc
     1081 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     1141 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     1201 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     1261 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     1321 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     1381 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     1441 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     1501 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     1561 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     1621 ggccgaggac tgccccgaag tccggcacct catgcacgcg gatttcggct ccaacaatgt
     1681 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     1741 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     1801 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     1861 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     1921 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     1981 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     2041 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aataatcagt
     2101 actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt
     2161 gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc
     2221 atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga
     2281 atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt gtttaaacga
     2341 gctcgaattc atcgatgata tcagatccac tagtggccta tgcggccgcg gatctgccgg
     2401 tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg
     2461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4381 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4441 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4501 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     4561 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     4621 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4681 accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca
     4741 tac
//
