LOCUS       sequence-367949-        4100 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_nat 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 4100)
  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 4100)
  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_nat
FEATURES             Location/Qualifiers
     source          1..4100
                     /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"
     gene            complement(973..2092)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(973..1170)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1176..1748)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1749..2092)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(2124..2143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2167..2435
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      2522..2608
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2700..2727
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(2805..2852)
                     /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(2865..3659)
                     /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(2976..2995)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3586..3605
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        3973..4047
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4052..4071
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg 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 gcttccaggg 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 atcatcgatg aattcgagct cgttttcgac actggatggc ggcgttagta
      961 tcgaatcgac agcagtatag cgaccagcat tcacatacga ttgacgcatg atattacttt
     1021 ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg cgaaaaaaaa tataaatcac
     1081 gctaacattt gattaaaata gaacaactac aatataaaaa aactatacaa atgacaagtt
     1141 cttgaaaaca agaatctttt tattgtcagt actgattagg ggcagggcat gctcatgtag
     1201 agcgcctgct cgccgtccga ggcggtgccg tcgtacaggg cggtgtccag gccgcagagg
     1261 gtgaacccca tccgccggta cgcgtggatc gccggtgcgt tgacgttggt gacctccagc
     1321 cagaggtgcc cggcgccccg ctcgcgggcg aactccgtcg cgagccccat caacgcgcgc
     1381 ccgaccccgt gcccccggtg ctccggggcg acctcgatgt cctcgacggt cagccggcgg
     1441 ttccagccgg agtacgagac gaccacgaag cccgccaggt cgccgtcgtc cccgtacgcg
     1501 acgaacgtcc gggagtccgg gtcgccgtcc tccccgtcgt ccgattcgtc gtccgattcg
     1561 tcgtcgggga acaccttggt caggggcggg tccaccggca cctcccgcag ggtgaagccg
     1621 tccccggtgg cggtgacgcg gaagacggtg tcggtggtga aggacccatc cagtgcctcg
     1681 atggcctcgg cgtcccccgg gacactggtg cggtaccggt aagccgtgtc gtcaagagtg
     1741 gtacccatgg ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa
     1801 aaggaagtat atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca
     1861 ggggcgcggc gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg
     1921 tctgcagcga ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg
     1981 caaatgatta tacatgggga tgtatgggct aaatgtacgg gcgacagtca catcatgccc
     2041 ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctgatta
     2101 ccctgttatc cctacactct cagtacaatc tgctctgatg ccgcatagtt aagccagccg
     2161 ggtaattctt tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt
     2221 gatgttttct ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga
     2281 ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt
     2341 tctgttcaaa agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc
     2401 gttcgaaact tctccgcagt gaaagataaa tgatcatttc cccgaaaagt gccacctgca
     2461 tcgatggccc ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     2521 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     2581 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     2641 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     2701 gaaggccatc ctgacggatg gcctttttgc gtggccagtg ccaagcttgc atgcagattg
     2761 cagcattaca cgtcttgagc gattgtgtag gctggagctg cttcgaagtt cctatacttt
     2821 ctagagaata ggaacttcgg aataggaact tcaagatccc cttattagaa gaactcgtca
     2881 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     2941 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     3001 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     3061 ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg
     3121 tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     3181 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg
     3241 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     3301 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     3361 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     3421 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     3481 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     3541 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     3601 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg
     3661 cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt
     3721 ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc
     3781 ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat
     3841 gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata
     3901 gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg
     3961 ttccgcttcc ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     4021 acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc tcggcgaatt
     4081 gggtgcggcc gcgtttgtca
//
