LOCUS       sequence-259656-        6144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Csy4-NLS under TEF promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6144)
  AUTHORS   Ferreira R, Skrekas C, Nielsen J, David F
  TITLE     Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation using 
            Csy4 in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2017 Nov 21. doi: 10.1021/acssynbio.7b00259.
  PUBMED    29161506
REFERENCE   2  (bases 1 to 6144)
  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: "ACS Synth 
            Biol. 2017 Nov 21. doi: 10.1021/acssynbio.7b00259."
COMMENT     Sequence Label: p413_Csy4NLS
FEATURES             Location/Qualifiers
     source          1..6144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     495..513
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(551..573)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     673..692
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        839..1026
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1027..1689
                     /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(1949..2404)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2086..2107)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2298..2317
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2530..2552
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2544..2561
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2545..2561
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2571..2590
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2571..2589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2608..2855)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2821..2839
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(3430..3450)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3477..3875)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     complement(3893..3913)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3893..3911)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3932..3948)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3932..3948)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3945..3967)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3956..3972
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3980..3986,3987..4004,4005..4010))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4025..4046
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4163..4180)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4334..4922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4414..4433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5093..5884,5885..5953))
                     /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     5716..5735
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5954..6058)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6088..6144,1..447)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa
       61 agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca
      121 acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta aatagatata
      181 cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca
      241 cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt
      301 agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt
      361 tctttaattt ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa
      421 attataatta tttttatagc acgtgatgaa aaggacccta agaaaccatt attatcatga
      481 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
      541 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
      601 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
      661 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataaa ttcccgtttt
      721 aagagcttgg tgagcgctag gagtcactgc caggtatcgt ttgaacacgg cattagtcag
      781 ggaagtcata acacagtcct ttcccgcaat tttctttttc tattactctt ggcctcctct
      841 agtacactct atattttttt atgcctcggt aatgattttc attttttttt ttccacctag
      901 cggatgactc tttttttttc ttagcgattg gcattatcac ataatgaatt atacattata
      961 taaagtaatg tgatttcttc gaagaatata ctaaaaaatg agcaggcaag ataaacgaag
     1021 gcaaagatga cagagcagaa agccctagta aagcgtatta caaatgaaac caagattcag
     1081 attgcgatct ctttaaaggg tggtccccta gcgatagagc actcgatctt cccagaaaaa
     1141 gaggcagaag cagtagcaga acaggccaca caatcgcaag tgattaacgt ccacacaggt
     1201 atagggtttc tggaccatat gatacatgct ctggccaagc attccggctg gtcgctaatc
     1261 gttgagtgca ttggtgactt acacatagac gaccatcaca ccactgaaga ctgcgggatt
     1321 gctctcggtc aagcttttaa agaggcccta ggggccgtgc gtggagtaaa aaggtttgga
     1381 tcaggatttg cgcctttgga tgaggcactt tccagagcgg tggtagatct ttcgaacagg
     1441 ccgtacgcag ttgtcgaact tggtttgcaa agggagaaag taggagatct ctcttgcgag
     1501 atgatcccgc attttcttga aagctttgca gaggctagca gaattaccct ccacgttgat
     1561 tgtctgcgag gcaagaatga tcatcaccgt agtgagagtg cgttcaaggc tcttgcggtt
     1621 gccataagag aagccacctc gcccaatggt accaacgatg ttccctccac caaaggtgtt
     1681 cttatgtagt gacaccgatt atttaaagct gcagcatacg atatatatac atgtgtatat
     1741 atgtatacct atgaatgtca gtaagtatgt atacgaacag tatgatactg aagatgacaa
     1801 ggtaatgcat cattctatac gtgtcattct gaacgaggcg cgctttcctt ttttcttttt
     1861 gctttttctt tttttttctc ttgaactcga cggatctatg cggtgtgaaa taccgcacag
     1921 atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc
     1981 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2041 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2101 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2161 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2221 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2281 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2341 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2401 gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     2461 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     2521 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     2581 cactataggg cgaattgggt accggccgca aattaaagcc ttcgagcgtc ccaaaacctt
     2641 ctcaagcaag gttttcagta taatgttaca tgcgtacacg cgtctgtaca gaaaaaaaag
     2701 aaaaatttga aatataaata acgttcttaa tactaacata actataaaaa aataaatagg
     2761 gacctagact tcaggttgtc taactccttc cttttcggtt agagcggatg tggggggagg
     2821 gcgtgaatgt aagcgtgaca taactaatta catgactcag aaccatggca cgaaacctcc
     2881 tttggacaac ccatagcagg tgaaccctcc ttcctcagcg gtcacttgca gaggtccgtg
     2941 cctgatgaac agcctaaagt gctgccctgt gctttgacta cgcaaggtaa caaaaggaag
     3001 atctagggca cgcgctactg tgtccggtat cctcttcctc gcctcctctt ctgacaggtc
     3061 atgtcttctc atcaatcttc tccttagacg ctcaggattg cttttggctt gtactctgga
     3121 aacctgtcta tacggcgtag ggtgtggtac gaccgcaggt tctccgaatt gcagatgatc
     3181 tcttagcccc tctaaccagg gacgggccaa caaggctctt aaatcgtcgg ctgatgcatg
     3241 tattctcagc ctctcaccta gacgactacg actctcatct aggtcaggga aactgacacc
     3301 aatacgatcc cccccttgcg ccactaaagc ctggtgtagt ttgccgaaaa gcacactcat
     3361 tagctgggcg ggtggaaact ctggatcagg tcttaatctg atgtccaaat agtgatccat
     3421 cccgggtcta acttttctct tcttctttgg ggctctagac atatttgtaa ttaaaactta
     3481 gattagattg ctatgctttc tttctaatga gcaagaagta aaaaaagttg taatagaaca
     3541 agaaaaatga aactgaaact tgagaaattg aagaccgttt attaacttaa atatcaatgg
     3601 gaggtcatcg aaagagaaaa aaatcaaaaa aaaaattttc aagaaaaaga aacgtgataa
     3661 aaatttttat tgcctttttc gacgaagaaa aagaaacgag gcggtctctt ttttcttttc
     3721 caaaccttta gtacgggtaa ttaacgacac cctagaggaa gaaagagggg aaatttagta
     3781 tgctgtgctt gggtgttttg aagtggtacg gcgatgcgcg gagtccgaga aaatctggaa
     3841 gagtaaaaaa ggagtagaaa cattttgaag ctatgagctc cagcttttgt tccctttagt
     3901 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3961 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4021 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4081 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4141 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4201 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4261 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4321 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4381 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4441 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4501 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4561 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4621 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4681 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4741 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4801 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4861 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4921 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4981 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5041 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5101 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5161 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5221 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5281 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5341 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5401 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5461 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5521 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5581 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5641 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5701 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5761 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5821 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5881 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5941 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6001 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6061 catttccccg aaaagtgcca cctgacggat cgcttgcctg taacttacac gcgcctcgta
     6121 tcttttaatg atggaataat ttgg
//
