LOCUS       sequence-371408-        3634 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_mic recombines in vivo with
            a PCR product from pEasyG2_zeo/nat/hph..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3634)
  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 3634)
  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_mic
FEATURES             Location/Qualifiers
     source          1..3634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(943..1531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1023..1042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        1594..1668
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1673..1692
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1745..1763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      1804..3146
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    2136..2183
                     /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        3173..3277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3278..3346,3347..3634,1..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(3496..3515)
                     /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 acccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      661 atcaaaggat cttctcttta aaagataatg tatgattatg ctttcactca tatttataca
      721 gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt tgaagtacaa
      781 ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt ttcacaccct
      841 acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt ggtgcgcatg
      901 tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tctttccata ggctccgccc
      961 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1021 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1081 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1141 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1201 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1261 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1321 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1381 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1441 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1501 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1561 tgacgctcag tggaacgaaa actcacgtta agggttttag agctagaaat agcaagttaa
     1621 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtggt gctttttttg
     1681 ttttttatgt ctcggcgaat tgggtgcggc cgcgtttgtc acctttcata ggcctgacga
     1741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1801 tatgatccaa tatcaaagga aatgatagca ttgaaggatg agactaatcc aattgaggag
     1861 tggcagcata tagaacagct aaagggtagt gctgaaggaa gcatacgata ccccgcatgg
     1921 aatgggataa tatcacagga ggtactagac tacctttcat cctacataaa tagacgcata
     1981 taagtacgca tttaagcata aacacgcact atgccgttct tctcatgtat atatatatac
     2041 aggcaacacg cagatatagg tgcgacgtga acagtgagct gtatgtgcgc agctcgcgtt
     2101 gcattttcgg aagcgctcgt tttcggaaac gctttgaagt tcctattccg aagttcctat
     2161 tctctagaaa gtataggaac ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc
     2221 actttcaaaa aaccaaaaac gcaccggact gtaacgagct actaaaatat tgcgaatacc
     2281 gcttccacaa acattgctca aaagtatctc tttgctatat atctctgtgc tatatcccta
     2341 tataacctac ccatccacct ttcgctcctt gaacttgcat ctaaactcga cctctacatt
     2401 ttttatgttt atctctagta ttactcttta gacaaaaaaa ttgtagtaag aactattcat
     2461 agagtgaatc gaaaacaata cgaaaatgta aacatttcct atacgtagta tatagagaca
     2521 aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt
     2581 ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc
     2641 ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc
     2701 tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta
     2761 cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat
     2821 ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga
     2881 agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat
     2941 gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa
     3001 aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta
     3061 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct
     3121 acaaaatgaa gcacagatgc ttcgttcagg tggcactttt cggggaaatg tgcgcggaac
     3181 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3241 ctgataaatg cttcaataat attgaaaaag gaatagtatg agtattcaac atttccgtgt
     3301 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3361 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3421 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3481 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3541 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3601 aaagcatctt acggatggca tgacagtaag agaa
//
