LOCUS       sequence-367571-        7185 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 7185)
  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 7185)
  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_hph
FEATURES             Location/Qualifiers
     source          1..7185
                     /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..3264)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1689..1886)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1892..2920)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTSPRA
                     KE"
     promoter        complement(2921..3264)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(3296..3315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3339..3607
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      3694..3780
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3872..3899
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(3977..4024)
                     /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(4037..4831)
                     /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(4148..4167)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4758..4777
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        5145..5219
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5224..5243
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(5296..5314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      5355..6697
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5687..5734
                     /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        6724..6828
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6829..6897,6898..7185,1..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(7047..7066)
                     /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 attattcctt tgccctcgga ctagtgctgg
     1921 ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc
     1981 ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc
     2041 atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt
     2101 ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat
     2161 gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga
     2221 agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg
     2281 ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt
     2341 gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg
     2401 acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg
     2461 cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga
     2521 acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catggcctcc gcgaccggct
     2581 gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc
     2641 gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact tccggaatcg
     2701 ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc
     2761 agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt
     2821 cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact
     2881 tctcgacaga cgtcgcggtg agttcaggct ttttacccat ggttgtttat gttcggatgt
     2941 gatgtgagaa ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag
     3001 tggcaaatcc taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc
     3061 gtctgtgagg ggagcgtttc cctgctcgca ggtctgcagc gaggagccgt aatttttgct
     3121 tcgcgccgtg cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg
     3181 ctaaatgtac gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag
     3241 gagggtattc tgggcctcca tgtcgctgat taccctgtta tccctacact ctcagtacaa
     3301 tctgctctga tgccgcatag ttaagccagc cgggtaattc tttgaaaaga taatgtatga
     3361 ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta tatacaaggt
     3421 gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg ggctagcggt
     3481 aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg gtcaaacgct
     3541 gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca gtgaaagata
     3601 aatgatcatt tccccgaaaa gtgccacctg catcgatggc ccccgatggt agtgtggggt
     3661 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3721 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3781 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     3841 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     3901 gcgtggccag tgccaagctt gcatgcagat tgcagcatta cacgtcttga gcgattgtgt
     3961 aggctggagc tgcttcgaag ttcctatact ttctagagaa taggaacttc ggaataggaa
     4021 cttcaagatc cccttattag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     4081 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     4141 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     4201 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     4261 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     4321 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     4381 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     4441 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     4501 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     4561 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     4621 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     4681 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     4741 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     4801 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     4861 cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     4921 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg
     4981 tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc
     5041 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     5101 agcaccgttt ctgcggactg gctttctacg tgttccgctt ccttgtttta gagctagaaa
     5161 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgg
     5221 tgcttttttt gttttttatg tctcggcgaa ttgggtgcgg ccgcgtttgt cacctttcat
     5281 aggcctgacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5341 tggtttctta gtatgatcca atatcaaagg aaatgatagc attgaaggat gagactaatc
     5401 caattgagga gtggcagcat atagaacagc taaagggtag tgctgaagga agcatacgat
     5461 accccgcatg gaatgggata atatcacagg aggtactaga ctacctttca tcctacataa
     5521 atagacgcat ataagtacgc atttaagcat aaacacgcac tatgccgttc ttctcatgta
     5581 tatatatata caggcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg
     5641 cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc
     5701 gaagttccta ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc
     5761 tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata
     5821 ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg
     5881 ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg
     5941 acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa
     6001 gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt
     6061 atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac
     6121 gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg
     6181 atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag
     6241 tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct
     6301 taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga
     6361 aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag
     6421 aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt
     6481 tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt
     6541 tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt
     6601 tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc
     6661 atttttgttc tacaaaatga agcacagatg cttcgttcag gtggcacttt tcggggaaat
     6721 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6781 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaatagtat gagtattcaa
     6841 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6901 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     6961 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7021 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7081 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     7141 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaa
//
