LOCUS       sequence-367565-        6543 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 6543)
  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 6543)
  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_zeo
FEATURES             Location/Qualifiers
     source          1..6543
                     /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)."
     terminator      complement(1689..1886)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(2279..2622)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(2654..2673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2697..2965
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      3052..3138
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3230..3257
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(3335..3382)
                     /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(3395..4189)
                     /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(3506..3525)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4116..4135
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_RNA        4503..4577
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4582..4601
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(4654..4672)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4713..6055
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5045..5092
                     /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        6082..6186
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6187..6255,6256..6543,1..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(6405..6424)
                     /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 atcatgagat gcctgcaagc aattcgttct
     1921 gtatcaggcg caggagcgtc ccgtccgggt cgaccaaagc ggccatcgtg cctccccact
     1981 cctgcagttc gggggcatgg atgcgcggat agccgctgct ggtttcctgg atgccgacgg
     2041 atttgcactg ccggtagaac tccgcgaggt cgtccagcct caggcagcag ctgaaccaac
     2101 tcgcgagggg atcgagcccg gggtgggcga agaactccag catgagatcc ccgcgctgga
     2161 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     2221 cggtgggatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atacccatgg
     2281 ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa aaggaagtat
     2341 atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca ggggcgcggc
     2401 gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg tctgcagcga
     2461 ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta
     2521 tacatgggga tgtatgggct aaatgtacgg gcgacagtca catcatgccc ctgagctgcg
     2581 cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctgatta ccctgttatc
     2641 cctacactct cagtacaatc tgctctgatg ccgcatagtt aagccagccg ggtaattctt
     2701 tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt gatgttttct
     2761 ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga ttttgtagtg
     2821 ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt tctgttcaaa
     2881 agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact
     2941 tctccgcagt gaaagataaa tgatcatttc cccgaaaagt gccacctgca tcgatggccc
     3001 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     3061 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3121 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     3181 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     3241 ctgacggatg gcctttttgc gtggccagtg ccaagcttgc atgcagattg cagcattaca
     3301 cgtcttgagc gattgtgtag gctggagctg cttcgaagtt cctatacttt ctagagaata
     3361 ggaacttcgg aataggaact tcaagatccc cttattagaa gaactcgtca agaaggcgat
     3421 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     3481 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     3541 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     3601 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     3661 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     3721 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     3781 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     3841 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     3901 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     3961 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     4021 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     4081 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     4141 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     4201 ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     4261 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     4321 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac
     4381 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc
     4441 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg ttccgcttcc
     4501 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     4561 gtggcaccga gtcggtggtg ctttttttgt tttttatgtc tcggcgaatt gggtgcggcc
     4621 gcgtttgtca cctttcatag gcctgacgaa agggcctcgt gatacgccta tttttatagg
     4681 ttaatgtcat gataataatg gtttcttagt atgatccaat atcaaaggaa atgatagcat
     4741 tgaaggatga gactaatcca attgaggagt ggcagcatat agaacagcta aagggtagtg
     4801 ctgaaggaag catacgatac cccgcatgga atgggataat atcacaggag gtactagact
     4861 acctttcatc ctacataaat agacgcatat aagtacgcat ttaagcataa acacgcacta
     4921 tgccgttctt ctcatgtata tatatataca ggcaacacgc agatataggt gcgacgtgaa
     4981 cagtgagctg tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg
     5041 ctttgaagtt cctattccga agttcctatt ctctagaaag tataggaact tcagagcgct
     5101 tttgaaaacc aaaagcgctc tgaagacgca ctttcaaaaa accaaaaacg caccggactg
     5161 taacgagcta ctaaaatatt gcgaataccg cttccacaaa cattgctcaa aagtatctct
     5221 ttgctatata tctctgtgct atatccctat ataacctacc catccacctt tcgctccttg
     5281 aacttgcatc taaactcgac ctctacattt tttatgttta tctctagtat tactctttag
     5341 acaaaaaaat tgtagtaaga actattcata gagtgaatcg aaaacaatac gaaaatgtaa
     5401 acatttccta tacgtagtat atagagacaa aatagaagaa accgttcata attttctgac
     5461 caatgaagaa tcatcaacgc tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg
     5521 tatagaatat aatcggggat gcctttatct tgaaaaaatg cacccgcagc ttcgctagta
     5581 atcagtaaac gcgggaagtg gagtcaggct ttttttatgg aagagaaaat agacaccaaa
     5641 gtagccttct tctaacctta acggacctac agtgcaaaaa gttatcaaga gactgcatta
     5701 tagagcgcac aaaggagaaa aaaagtaatc taagatgctt tgttagaaaa atagcgctct
     5761 cgggatgcat ttttgtagaa caaaaaagaa gtatagattc tttgttggta aaatagcgct
     5821 ctcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg aaaaattagc
     5881 gctctcgcgt tgcatttttg ttttacaaaa atgaagcaca gattcttcgt tggtaaaata
     5941 gcgctttcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa
     6001 ttagcgctct cgcgttgcat ttttgttcta caaaatgaag cacagatgct tcgttcaggt
     6061 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6121 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6181 aatagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6241 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6301 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6361 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6421 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6481 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6541 gaa
//
