LOCUS       sequence-266844-        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast intergration vector for integration of dual reporter system at
            the URA3 locus. Dual reporter system contains constitutive mCherry 
            and constitutive eGFP flanked by iCas9 sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 7458)
  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"; journalName: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pMG
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        317..503
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             504..1163
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(1423..1878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1560..1581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1772..1791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1961..2126)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2047..2071
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2270..2980)
                     /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(2548..2567)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2817..2835
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        complement(2998..3404)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3948..4346
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4364..5080
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(5468..5485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5639..6227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5719..5738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6398..7189,7190..7258))
                     /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     7021..7040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7259..7363)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7431..7449
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata
      421 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc
      481 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa
      541 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact
      601 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga
      661 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt
      721 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca
      781 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag
      841 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag
      901 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag
      961 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta
     1021 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca
     1081 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct
     1141 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat
     1201 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat
     1261 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt
     1321 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt
     1381 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata
     1441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     1501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     1561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1861 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     1921 gatcggtgcg ggcctcttcg ctattacgcc agctgaattg gagcgacctc atgctatacc
     1981 tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt tcgttttaaa
     2041 acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta tttaataata
     2101 aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta tctaccaacg
     2161 atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc gacaaccttg
     2221 attggagact tgaccaaacc tctggcgaag aattgttaat taagagctct tacttgtaca
     2281 gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt tccacgatgg
     2341 tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag gcgccgggca
     2401 gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag tggccgccgt
     2461 ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc
     2521 gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga
     2581 agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg gaggagtcct
     2641 gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct
     2701 cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg
     2761 agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccacccttgg
     2821 tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga
     2881 tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct
     2941 tgatgatggc catgttatcc tcctcgccct tgctcaccat tttaataact agtaaaactt
     3001 agattagatt gctatgcttt ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac
     3061 aagaaaaatg aaactgaaac ttgagaaatt gaagaccgtt tattaactta aatatcaatg
     3121 ggaggtcatc gaaagagaaa aaaatcaaaa aaaaaatttt caagaaaaag aaacgtgata
     3181 aaaattttta ttgccttttt cgacgaagaa aaagaaacga ggcggtctct tttttctttt
     3241 ccaaaccttt agtacgggta attaacgaca ccctagagga agaaagaggg gaaatttagt
     3301 atgctgtgct tgggtgtttt gaagtggtac ggcgatgcgc ggagtccgag aaaatctgga
     3361 agagtaaaaa aggagtagaa acattttgaa gctatggtgt ggcggccgct ttgatgttag
     3421 cagaattgtc atgcaagggc tccctagcta ctggagaata tactaagggt actgttgaca
     3481 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa
     3541 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag
     3601 acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacaagg cctgggccct
     3661 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
     3721 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
     3781 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
     3841 ggtgaattct ccgatccatc ccccaggctt gcactcgtac gttcgaaata ttataaatta
     3901 tcagacagac catactccag atgggggatg gctaggtgaa ttcacaccat agcttcaaaa
     3961 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac
     4021 ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt
     4081 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg
     4141 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt tttttttgat
     4201 ttttttctct ttcgatgacc tcccattgat atttaagtta ataaacggtc ttcaatttct
     4261 caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt gctcattaga
     4321 aagaaagcat agcaatctaa tctaagtttt ggatcctatt aaaatgtcta aaggtgaaga
     4381 attattcact ggtgttgtcc caattttggt tgaattagat ggtgatgtta atggtcacaa
     4441 attttctgtc tccggtgaag gtgaaggtga tgctacttac ggtaaattga ccttaaaatt
     4501 tatttgtact actggtaaat tgccagttcc atggccaacc ttagtcacta ctttcggtta
     4561 tggtgttcaa tgttttgcta gatacccaga tcatatgaaa caacatgact ttttcaagtc
     4621 tgccatgcca gaaggttatg ttcaagaaag aactattttt ttcaaagatg acggtaacta
     4681 caagaccaga gctgaagtca agtttgaagg tgatacctta gttaatagaa tcgaattaaa
     4741 aggtattgat tttaaagaag atggtaacat tttaggtcac aaattggaat acaactataa
     4801 ctctcacaat gtttacatca tggctgacaa acaaaagaat ggtatcaaag ttaacttcaa
     4861 aattagacac aacattgaag atggttctgt tcaattagct gaccattatc aacaaaatac
     4921 tccaattggt gatggtccag tcttgttacc agacaaccat tacttatcca ctcaatctgc
     4981 cttatccaaa gatccaaacg aaaagagaga ccacatggtc ttgttagaat ttgttactgc
     5041 tgctggtatt acccatggta tggatgaatt gtacaaataa ctcgagtaag cttggtaccg
     5101 cggctagcta agatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     5161 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     5221 tctttttttt ctgtacagac gcgttccgat ccatccccca ggcttgcact cgtacgttcg
     5281 aaatattata aattatcaga cagaccatac tccagatggg ggatggctag gtacgcgtgt
     5341 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcc
     5401 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5461 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5521 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5581 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5641 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5701 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5761 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5821 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5881 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5941 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6001 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6061 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6121 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6181 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6241 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6301 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6361 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6421 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6481 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6541 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6601 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6661 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6721 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6781 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6841 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6901 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6961 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7021 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7081 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7141 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7201 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7261 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7321 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7381 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     7441 tcacgaggcc ctttcgtc
//
