LOCUS       sequence-273626-        7457 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 7457)
  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 7457)
  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..7457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        260..658
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             676..1392
                     /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(1780..1797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1951..2539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2031..2050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2710..3501,3502..3570))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFSMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3333..3352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3571..3675)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3743..3761
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3799..3821)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3921..3940
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4087..4274
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             4275..4937
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(5197..5652)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5334..5355)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5546..5565
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5730..5895)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5816..5840
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(6039..6749)
                     /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(6317..6336)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6586..6604
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        complement(6767..7173)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 gttaagccgc taaaggcatt atccgccaag tacaattttt tactcttcga agacagaaaa
       61 tttgctgaca ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca
      121 gaatgggcag acattacgaa tgcacacggt gtggtgaatt ctccgatcca tcccccaggc
      181 ttgcactcgt acgttcgaaa tattataaat tatcagacag accatactcc agatggggga
      241 tggctaggtg aattcacacc atagcttcaa aatgtttcta ctcctttttt actcttccag
      301 attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac agcgtactaa
      361 atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag
      421 aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca
      481 cgtttctttt tcttgaaaat tttttttttg atttttttct ctttcgatga cctcccattg
      541 atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct
      601 attacaactt tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaagtt
      661 ttggatccta ttaaaatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg
      721 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt
      781 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt
      841 ccatggccaa ccttagtcac tactttcggt tatggtgttc aatgttttgc gagataccca
      901 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa
      961 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa
     1021 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac
     1081 attttaggtc acaaattgga atacaactat aactctcaca atgtttacat catggctgac
     1141 aaacaaaaga atggtatcaa agttaacttc aaaattagac acaacattga agatggttct
     1201 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta
     1261 ccagacaacc attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga
     1321 gaccacatgg tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa
     1381 ttgtacaaat aactcgagta agcttggtac cgcggctagc taagatccgc tctaaccgaa
     1441 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     1501 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgttccg
     1561 atccatcccc caggcttgca ctcgtacgtt cgaaatatta taaattatca gacagaccat
     1621 actccagatg ggggatggct aggtacgcgt gtacgcatgt aacattatac tgaaaacctt
     1681 gcttgagaag gttttgggac gctcgaagat ccagctgcat taatgaatcg gccaacgcgc
     1741 ggggagaggc ggtttgcgta ttgggcgctg ttccgcttcc tcgctcactg actcgctgcg
     1801 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1861 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1921 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1981 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2041 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2101 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2161 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2221 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2281 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2341 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2401 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2461 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2521 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2581 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2641 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2701 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2761 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2821 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2881 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     2941 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3001 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3061 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3121 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3181 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3241 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3301 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3361 aaaagtgctc atcattgaaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3421 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3481 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3541 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3601 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3661 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     3721 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     3781 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3841 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3901 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaatt
     3961 cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt gaacacggca
     4021 ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta ttactcttgg
     4081 cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat tttttttttt
     4141 ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat aatgaattat
     4201 acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag caggcaagat
     4261 aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca aatgaaacca
     4321 agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac tcgatcttcc
     4381 cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg attaacgtcc
     4441 acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat tccggctggt
     4501 cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc actgaagact
     4561 gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt ggagtaaaaa
     4621 ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg gtagatcttt
     4681 cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta ggagatctct
     4741 cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga attaccctcc
     4801 acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg ttcaaggctc
     4861 ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt ccctccacca
     4921 aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat atatatacat
     4981 gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta tgatactgaa
     5041 gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg ctttcctttt
     5101 ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg gtgtgaaata
     5161 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt
     5221 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5281 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5341 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     5401 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5461 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5521 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5581 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     5641 tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5701 ggcctcttcg ctattacgcc actgaattgg agcgacctca tgctatacct gagaaagcaa
     5761 cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc
     5821 actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa
     5881 tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctaccaacga tttgaccctt
     5941 ttccatcttt tcgtaaattt ctggcaaggt agacaagccg acaaccttga ttggagactt
     6001 gaccaaacct ctggcgaaga attgttaatt aagagctctt acttgtacag ctcgtccatg
     6061 ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg
     6121 ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg cgccgggcag ctgcacgggc
     6181 ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc
     6241 agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag
     6301 gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa gttggtgccg
     6361 cgcagcttca ccttgtagat gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc
     6421 accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac
     6481 agcttcaagt agtcggggat gtcggcgggg tgcttcacgt aggccttgga gccgtacatg
     6541 aactgagggg acaggatgtc ccaggcgaag ggcagggggc cacccttggt caccttcagc
     6601 ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg
     6661 tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt gatgatggcc
     6721 atgttatcct cctcgccctt gctcaccatt ttaataacta gtaaaactta gattagattg
     6781 ctatgctttc tttctaatga gcaagaagta aaaaaagttg taatagaaca agaaaaatga
     6841 aactgaaact tgagaaattg aagaccgttt attaacttaa atatcaatgg gaggtcatcg
     6901 aaagagaaaa aaatcaaaaa aaaaattttc aagaaaaaga aacgtgataa aaatttttat
     6961 tgcctttttc gacgaagaaa aagaaacgag gcggtctctt ttttcttttc caaaccttta
     7021 gtacgggtaa ttaacgacac cctagaggaa gaaagagggg aaatttagta cgctgtgctt
     7081 gggtgttttg aagtggtacg gcgatgcgcg gagtccgaga aaatctggaa gagtaaaaaa
     7141 ggagtagaaa cattttgaag ctatggtgtg gcggccgctt tgatgttagc agaattgtca
     7201 tgcaagggct ccctagctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     7261 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     7321 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt
     7381 caacagtata gaaccgtgga tgatgtggtc tctacaaggc ctgggccctc ttgactgatt
     7441 tttccatgga gggcaca
//
