LOCUS       sequence-39187-e        4447 bp    DNA     circular SYN 14-APR-2025
DEFINITION  genomic GFP tagging in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4447)
  AUTHORS   Gadaleta MC, Iwasaki O, Noguchi C, Noma K, Noguchi E
  TITLE     New vectors for epitope tagging and gene disruption in 
            Schizosaccharomyces pombe.
  JOURNAL   Biotechniques. 2013 Nov;55(5):257-63. doi: 10.2144/000114100.
  PUBMED    24215641
REFERENCE   2  (bases 1 to 4447)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Biotechniques"; date: "2013-11"; volume: "55"; issue: "5"; pages: 
            "257-63"
COMMENT     Sequence Label: pFA6a-GFP-bleMX6
FEATURES             Location/Qualifiers
     source          1..4447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..773
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(90..118)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     713..733
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      797..984
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(852..876)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1059..1980
                     /label=bleMX6
                     /note="yeast selectable marker conferring phleomycin 
                     resistance"
     promoter        1059..1402
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1403..1777
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      1783..1980
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2084..2103)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2085..2103)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2190..2207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2361..2949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2441..2460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3120..3911,3912..3980))
                     /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     3743..3762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3981..4085)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4153..4171
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4209..4231)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4331..4350
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4431..4447,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4431..4447,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 acagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
      121 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
      181 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
      241 tcactacttt cacttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc
      301 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
      361 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
      421 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
      481 tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa
      541 tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc
      601 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
      661 tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
      721 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaatagggcg
      781 cgccacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
      841 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
      901 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc
      961 tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatctg
     1021 tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc
     1081 tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt
     1141 agcccataca tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc
     1201 gaagcaaaaa ttacggctcc tcgctgcaga cctgcgagca gggaaacgct cccctcacag
     1261 acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc
     1321 cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca
     1381 tcacatccga acataaacaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
     1441 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
     1501 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
     1561 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
     1621 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc
     1681 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
     1741 caactgcgtg cacttcgtgg ccgaggagca ggactgatca gtactgacaa taaaaagatt
     1801 cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa
     1861 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     1921 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     1981 ctgtcgattc gatactaacg ccgccatcca gtttaaacga gctcgaattc atcgatgata
     2041 tcagatccac tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta
     2101 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc
     2161 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2221 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2281 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2341 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2401 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2461 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2521 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     2581 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2641 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2701 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2761 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     2821 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2881 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2941 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3001 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3061 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3121 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3181 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3241 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3301 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3361 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3421 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3481 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3541 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3601 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3661 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3721 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3781 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3841 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3901 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3961 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4021 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4081 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4141 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     4201 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4261 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     4321 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg
     4381 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
     4441 cactata
//
