LOCUS       sequence-367561-        6729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template to generate via PCR a single gRNA for expression in S. 
            cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6729)
  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 6729)
  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: pEasyG1_nat
FEATURES             Location/Qualifiers
     source          1..6729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      675..1263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1164..1183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1417..1434
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1521..1540
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1521..1539
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1593..1626)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(1689..2808)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(1689..1886)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1892..2464)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(2465..2808)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(2840..2859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2883..3151
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      3238..3324
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3416..3443
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(3521..3568)
                     /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(3581..4375)
                     /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(3692..3711)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4302..4321
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        4689..4763
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4768..4787
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(4840..4858)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4899..6241
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5231..5278
                     /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)."
     promoter        6268..6372
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6373..6441,6442..6729,1..504)
                     /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     complement(6591..6610)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
       61 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      121 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      181 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      241 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      301 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      361 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      421 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      481 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
      541 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
      601 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      661 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      721 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      781 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      841 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      901 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      961 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1021 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1081 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1141 gagcgcacga gggagcttcc agtgggaaac gcctggtatc tttatagtcc tgtcgggttt
     1201 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1261 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1321 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1381 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1441 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg
     1501 ttaacctggc ttatcgaaat taatacgact cactataggg agaccggcag atccgcggcc
     1561 gcataggcca ctagtggatc tgatatcacc taataacttc gtatagcata cattatacga
     1621 agttatatta agggttctcg agagctcgtt ttcgacactg gatggcggcg ttagtatcga
     1681 atcgacagca gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc
     1741 gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta
     1801 acatttgatt aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg
     1861 aaaacaagaa tctttttatt gtcagtactg attaggggca gggcatgctc atgtagagcg
     1921 cctgctcgcc gtccgaggcg gtgccgtcgt acagggcggt gtccaggccg cagagggtga
     1981 accccatccg ccggtacgcg tggatcgccg gtgcgttgac gttggtgacc tccagccaga
     2041 ggtgcccggc gccccgctcg cgggcgaact ccgtcgcgag ccccatcaac gcgcgcccga
     2101 ccccgtgccc ccggtgctcc ggggcgacct cgatgtcctc gacggtcagc cggcggttcc
     2161 agccggagta cgagacgacc acgaagcccg ccaggtcgcc gtcgtccccg tacgcgacga
     2221 acgtccggga gtccgggtcg ccgtcctccc cgtcgtccga ttcgtcgtcc gattcgtcgt
     2281 cggggaacac cttggtcagg ggcgggtcca ccggcacctc ccgcagggtg aagccgtccc
     2341 cggtggcggt gacgcggaag acggtgtcgg tggtgaagga cccatccagt gcctcgatgg
     2401 cctcggcgtc ccccgggaca ctggtgcggt accggtaagc cgtgtcgtca agagtggtac
     2461 ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg
     2521 aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg
     2581 cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct cgcaggtctg
     2641 cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa
     2701 tgattataca tggggatgta tgggctaaat gtacgggcga cagtcacatc atgcccctga
     2761 gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgc tgattaccct
     2821 gttatcccta cactctcagt acaatctgct ctgatgccgc atagttaagc cagccgggta
     2881 attctttgaa aagataatgt atgattatgc tttcactcat atttatacag aaacttgatg
     2941 ttttctttcg agtatataca aggtgattac atgtacgttt gaagtacaac tctagatttt
     3001 gtagtgccct cttgggctag cggtaaaggt gcgcattttt tcacacccta caatgttctg
     3061 ttcaaaagat tttggtcaaa cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc
     3121 gaaacttctc cgcagtgaaa gataaatgat catttccccg aaaagtgcca cctgcatcga
     3181 tggcccccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     3241 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3301 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     3361 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     3421 gccatcctga cggatggcct ttttgcgtgg ccagtgccaa gcttgcatgc agattgcagc
     3481 attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta tactttctag
     3541 agaataggaa cttcggaata ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa
     3601 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
     3661 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
     3721 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt
     3781 ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg
     3841 gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt
     3901 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
     3961 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
     4021 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc
     4081 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag
     4141 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt
     4201 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
     4261 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
     4321 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa
     4381 acgatcctca tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg
     4441 gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag
     4501 ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa
     4561 gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc
     4621 agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc
     4681 gcttccttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     4741 gaaaaagtgg caccgagtcg gtggtgcttt ttttgttttt tatgtctcgg cgaattgggt
     4801 gcggccgcgt ttgtcacctt tcataggcct gacgaaaggg cctcgtgata cgcctatttt
     4861 tataggttaa tgtcatgata ataatggttt cttagtatga tccaatatca aaggaaatga
     4921 tagcattgaa ggatgagact aatccaattg aggagtggca gcatatagaa cagctaaagg
     4981 gtagtgctga aggaagcata cgataccccg catggaatgg gataatatca caggaggtac
     5041 tagactacct ttcatcctac ataaatagac gcatataagt acgcatttaa gcataaacac
     5101 gcactatgcc gttcttctca tgtatatata tatacaggca acacgcagat ataggtgcga
     5161 cgtgaacagt gagctgtatg tgcgcagctc gcgttgcatt ttcggaagcg ctcgttttcg
     5221 gaaacgcttt gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcag
     5281 agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc
     5341 ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt gctcaaaagt
     5401 atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc cacctttcgc
     5461 tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc tagtattact
     5521 ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa
     5581 atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg ttcataattt
     5641 tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat gcgcaatcca
     5701 catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg
     5761 ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga gaaaatagac
     5821 accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta tcaagagact
     5881 gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag
     5941 cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg ttggtaaaat
     6001 agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa
     6061 attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt cttcgttggt
     6121 aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     6181 gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca gatgcttcgt
     6241 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6301 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6361 aaaaggaata gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6421 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6481 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6541 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6601 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6661 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6721 gtaagagaa
//
