LOCUS       sequence-358285-        6164 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression vector for gRNA directed against CAN1 ORF..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6164)
  AUTHORS   Shively CA, Dong F, Mitra RD
  TITLE     A Suite of New Strain Construction Vectors for Gene Expression 
            Knockdown in Budding Yeast.
  JOURNAL   ACS Synth Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547.
  PUBMED    36650116
REFERENCE   2  (bases 1 to 6164)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547."
COMMENT     Sequence Label: CAN1 gRNA HIS
FEATURES             Location/Qualifiers
     source          1..6164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..1369
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    359..406
                     /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        1396..1500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1501..1569,1570..2361)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGVS
                     LIKHW"
     primer_bind     complement(1719..1738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2532..3120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3021..3040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3274..3291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3408..3429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3444..3449,3450..3467,3468..3474)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3482..3498
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3487..3509
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3506..3522
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3506..3522
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3541..3561
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3543..3561
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3579..3847
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3868..3942
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3947..3966
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      3972..4219
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4237..4256)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4238..4256)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4266..4283)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4266..4282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4275..4297)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4423..4878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4510..4529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4720..4741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5049..5235
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             5236..5898
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(6132..6150)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcatgataat aatggtttct tagtatgatc caatatcaaa ggaaatgata gcattgaagg
       61 atgagactaa tccaattgag gagtggcagc atatagaaca gctaaagggt agtgctgaag
      121 gaagcatacg ataccccgca tggaatggga taatatcaca ggaggtacta gactaccttt
      181 catcctacat aaatagacgc atataagtac gcatttaagc ataaacacgc actatgccgt
      241 tcttctcatg tatatatata tacaggcaac acgcagatat aggtgcgacg tgaacagtga
      301 gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct cgttttcgga aacgctttga
      361 agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcagag cgcttttgaa
      421 aaccaaaagc gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg actgtaacga
      481 gctactaaaa tattgcgaat accgcttcca caaacattgc tcaaaagtat ctctttgcta
      541 tatatctctg tgctatatcc ctatataacc tacccatcca cctttcgctc cttgaacttg
      601 catctaaact cgacctctac attttttatg tttatctcta gtattactct ttagacaaaa
      661 aaattgtagt aagaactatt catagagtga atcgaaaaca atacgaaaat gtaaacattt
      721 cctatacgta gtatatagag acaaaataga agaaaccgtt cataattttc tgaccaatga
      781 agaatcatca acgctatcac tttctgttca caaagtatgc gcaatccaca tcggtataga
      841 atataatcgg ggatgccttt atcttgaaaa aatgcacccg cagcttcgct agtaatcagt
      901 aaacgcggga agtgaagtca ggcttttttt atggaagaga aaatagacac caaagtagcc
      961 ttcttctaac cttaacggac ctacagtgca aaaagttatc aagagactgc attatagagc
     1021 gcacaaagga gaaaaaaagt aatctaagat gctttgttag aaaaatagcg ctctcgggat
     1081 gcatttttgt agaacaaaaa agaagtatag attctttgtt ggtaaaatag cgctctcgcg
     1141 ttgcatttct gttctgtaaa aatgcagctc agattctttg tttgaaaaat tagcgctctc
     1201 gcgttgcatt tttgttttac aaaaatgaag cacagattct tcgttggtaa aatagcgctt
     1261 tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga aaaattagcg
     1321 ctctcgcgtt gcatttttgt tctacaaaat gaagcacaga tgcttcgttc aggtggcact
     1381 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1441 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1501 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1561 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1621 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1681 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1741 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1801 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1861 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1921 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1981 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2041 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2101 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2161 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2221 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2281 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgtt
     2341 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2401 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2461 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2521 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2581 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2641 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2701 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2761 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2821 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2881 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2941 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3001 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3061 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3121 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3181 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3241 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3301 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     3361 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     3421 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     3481 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     3541 gcaattaacc ctcactaaag ggaacaaaag ctggagcttc tttgaaaaga taatgtatga
     3601 ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta tatacaaggt
     3661 gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg ggctagcggt
     3721 aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg gtcaaacgct
     3781 gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca gtgaaagata
     3841 aatgatcgat acgttctcta tggaggagtt ttagagctag aaatagcaag ttaaaataag
     3901 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tggtgctttt tttgtttttt
     3961 atgtcttcga gtcatgtaat tagttatgtc acgcttacgt tcacgccctc cccccacatc
     4021 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     4081 tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
     4141 cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     4201 ctcgaaggct ttaatttgcg gccggtaccc aattcgccct atagtgagtc gtattacgcg
     4261 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4381 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg
     4441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4561 gtcaagctct aaatcggggg ctccctttag ggttccgatt aagtgcttta cggcacctcg
     4621 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4681 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4741 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4801 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4861 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcaatatttc
     4921 ttcccgtttt aagagcttgg tgagcgctag gagtcactgc caggtatcgt ttgaacacgg
     4981 cattagtcag ggaagtcata acacagtcct ttcccgcaat tttctttttc tattactctt
     5041 ggcctcctct agtacactct atattttttt atgcctcggt aatgattttc attttttttt
     5101 tccacctagc ggatgactct ttttttttct tagcgattgg cattatcaca taatgaatta
     5161 tacattatat aaagtaatgt gatttcttcg aagaatatac taaaaaatga gcaggcaaga
     5221 taaacgaagg caaagatgac agagcagaaa gccctagtaa agcgtattac aaatgaaacc
     5281 aaaattcaga ttgcgatctc tttaaagggt ggtcccctag cgatagagca ctcgatcttc
     5341 ccagaaaaag aggcagaagc agtagcagaa caggccacac aatcgcaagt gattaacgtc
     5401 cacacaggta tagggtttct ggaccatatg atacatgctc tggccaagca ttccggctgg
     5461 tcgctaatcg ttgagtgcat tggtgactta cacatagacg accatcacac cactgaagac
     5521 tgcgggattg ctctcggtca agcttttaaa gaggccctag gggccgtgcg tggagtaaaa
     5581 aggtttggat caggatttgc gcctttggat gaggcacttt ccagagcggt ggtagatctt
     5641 tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa gggagaaagt aggagatctc
     5701 tcttgcgaga tgatcccgca ttttcttgaa agctttgcag aggctagcag aattaccctc
     5761 cacgttgatt gtctgcgagg caagaatgat catcaccgta gtgagagtgc gttcaaggct
     5821 cttgcggttg ccataagaga agccacctcg cccaatggta ccaacgatgt tccctccacc
     5881 aaaggtgttc ttatgtagtg acaccgatta tttaaagctg cagcatacga tatatataca
     5941 tgtgtatata tgtataccta tgaatgtcag taagtatgta tacgaacagt atgatactga
     6001 agatgacaag gtaatgcatc attctatacg tgtcattctg aacgaggcgc gctttccttt
     6061 tttctttttg ctttttcttt ttttttctct tgaactcgac ggatctatgc ggtgacgcgc
     6121 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatg
//
