LOCUS       sequence-264562-        5345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for SaCas9 gRNA cloning and expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5345)
  AUTHORS   Lian J, Schultz C, Cao M, HamediRad M, Zhao H
  TITLE     Multi-functional genome-wide CRISPR system for high throughput 
            genotype-phenotype mapping.
  JOURNAL   Nat Commun. 2019 Dec 19;10(1):5794. doi: 10.1038/s41467-019-13621-4.
  PUBMED    31857575
REFERENCE   2  (bases 1 to 5345)
  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"; journalName: "Nat 
            Commun."; date: "2019-12-19"; volume: "10(1):5794. doi"; pages: " 
            10.1038/s41467-019-13621-4"
COMMENT     Sequence Label: p426*-SaSgH
FEATURES             Location/Qualifiers
     source          1..5345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        1309..1577
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      1726..1745
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1783..1799)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1783..1799)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1796..1818)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1807..1823
                     /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)."
     promoter        complement(join(1831..1837,1838..1855,1856..1861))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1876..1897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2014..2031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2185..2773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2265..2284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2944..3735,3736..3804))
                     /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     3567..3586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3805..3909)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3936..5278
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4899..4946)
                     /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)."
     primer_bind     5318..5336
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtcgtttc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattaccctg tgctcgagtc tttgaaaaga
     1321 taatgtatga ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta
     1381 tatacaaggt gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg
     1441 ggctagcggt aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg
     1501 gtcaaacgct gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca
     1561 gtgaaagata aatgatcgga gaccgaggtc tccgttttag tactctgtaa ttttaggtat
     1621 gaggtagacg aaaattgtac ttatacctaa aattacagaa tctactaaaa caaggcaaaa
     1681 tgccgtgttt atctcgtcaa cttgttggcg agattttttt gatccttttt ttgtttttta
     1741 tgtctgagct ccctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt
     1801 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     1861 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     1921 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1981 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2041 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2101 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2161 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2221 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2281 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2341 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2401 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2461 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2521 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2581 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2641 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2701 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2761 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2821 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2881 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2941 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3001 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3061 ccccagtgct gcaatgatac cgcggctccc acgctcaccg gctccagatt tatcagcaat
     3121 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3181 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3241 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3301 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3361 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3421 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3481 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3541 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3601 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3661 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3721 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3781 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3841 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3901 ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg
     3961 tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt
     4021 ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat
     4081 ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg
     4141 catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac
     4201 ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta
     4261 gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact
     4321 gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa
     4381 agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca
     4441 agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca
     4501 gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt
     4561 ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc
     4621 tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa
     4681 aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat
     4741 atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag
     4801 cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag
     4861 tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag
     4921 aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg
     4981 caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc
     5041 tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt
     5101 atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat
     5161 tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc
     5221 actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat
     5281 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     5341 tcgtc
//
