LOCUS       sequence-379508-        3956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template to generate via PCR two gRNAs for expression in S. 
            cerevisiae. The PCR product from pEasyG2_zeo recombines in vivo with
            a PCR product from pEasyG2_mic..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3956)
  AUTHORS   Jacobus AP, Barreto JA, de Bem LS, Menegon YA, Fier I, Bueno JGR, 
            Dos Santos LV, Gross J
  TITLE     EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9
            Applications in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2022 Nov 18;11(11):3886-3891. doi: 
            10.1021/acssynbio.2c00348. Epub 2022 Oct 18.
  PUBMED    36257021
REFERENCE   2  (bases 1 to 3956)
  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: 
            "10.1021/acssynbio.2c00348"; journalName: "ACS Synth Biol"; date: 
            "2022-11-18- 18"; volume: "11"; issue: "11"; pages: "3886-3891"
COMMENT     Sequence Label: pEasyG2_zeo
FEATURES             Location/Qualifiers
     source          1..3956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     847..866
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        847..865
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(919..952)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(1015..1212)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(1605..1948)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(1980..1999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2023..2291
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      2378..2464
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2556..2583
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(2661..2708)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(2721..3515)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2832..2851)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3442..3461
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        3829..3903
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3908..3927
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
ORIGIN
        1 ttgagatcct ttttttctgc gggtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccagtg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttaa cctggcttat
      841 cgaaattaat acgactcact atagggagac cggcagatcc gcggccgcat aggccactag
      901 tggatctgat atcacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg
      961 ttctcgagag ctcgttttcg acactggatg gcggcgttag tatcgaatcg acagcagtat
     1021 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     1081 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     1141 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     1201 tttattgtca gtactgatca tgagatgcct gcaagcaatt cgttctgtat caggcgcagg
     1261 agcgtcccgt ccgggtcgac caaagcggcc atcgtgcctc cccactcctg cagttcgggg
     1321 gcatggatgc gcggatagcc gctgctggtt tcctggatgc cgacggattt gcactgccgg
     1381 tagaactccg cgaggtcgtc cagcctcagg cagcagctga accaactcgc gaggggatcg
     1441 agcccggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     1501 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt gggatcgaaa
     1561 tctcgtgatg gcaggttggg cgtcgcttgg tcggtcatac ccatggttgt ttatgttcgg
     1621 atgtgatgtg agaactgtat cctagcaaga ttttaaaagg aagtatatga aagaagaacc
     1681 tcagtggcaa atcctaacct tttatatttc tctacagggg cgcggcgtgg ggacaattca
     1741 acgcgtctgt gaggggagcg tttccctgct cgcaggtctg cagcgaggag ccgtaatttt
     1801 tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa tgattataca tggggatgta
     1861 tgggctaaat gtatgggcga cagtcacatc atgcccctga gctgcgcacg tcaagactgt
     1921 caaggagggt attctgggcc tccatgtcgc tgattaccct gttatcccta cactctcagt
     1981 acaatctgct ctgatgccgc atagttaagc cagccgggta attctttgaa aagataatgt
     2041 atgattatgc tttcactcat atttatacag aaacttgatg ttttctttcg agtatataca
     2101 aggtgattac atgtacgttt gaagtacaac tctagatttt gtagtgccct cttgggctag
     2161 cggtaaaggt gcgcattttt tcacacccta caatgttctg ttcaaaagat tttggtcaaa
     2221 cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc gaaacttctc cgcagtgaaa
     2281 gataaatgat catttccccg aaaagtgcca cctgcatcga tggcccccga tggtagtgtg
     2341 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     2401 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     2461 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     2521 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     2581 ttttgcgtgg ccagtgccaa gcttgcatgc agattgcagc attacacgtc ttgagcgatt
     2641 gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata
     2701 ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     2761 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     2821 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     2881 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     2941 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     3001 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     3061 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     3121 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     3181 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     3241 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     3301 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     3361 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     3421 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     3481 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     3541 tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     3601 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt
     3661 gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc
     3721 tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg
     3781 ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttgt tttagagcta
     3841 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3901 gtggtgcttt ttttgttttt tatgtctcgg cgaattgggt gcggccgcgt ttgtca
//
