LOCUS       sequence-264564-        5233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for LbCpf1 gRNA cloning and expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5233)
  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 5233)
  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*-LbSgH
FEATURES             Location/Qualifiers
     source          1..5233
                     /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      1614..1633
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1671..1687)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1671..1687)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1684..1706)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1695..1711
                     /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(1719..1725,1726..1743,1744..1749))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1764..1785
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1902..1919)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2073..2661)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2153..2172)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2832..3623,3624..3692))
                     /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     3455..3474
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3693..3797)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3824..5166
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4787..4834)
                     /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     5206..5224
                     /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 aatgatcaat ttctactaag tgtagatgga gaccgaggtc tccttttttt
     1621 gttttttatg tctgagctcc ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     1681 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1741 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1801 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1861 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1981 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2041 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2101 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2161 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2221 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2281 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2341 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2401 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2461 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2521 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2581 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2641 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2701 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2761 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2821 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2881 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2941 ccatctggcc ccagtgctgc aatgataccg cggctcccac gctcaccggc tccagattta
     3001 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3061 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3121 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3181 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3241 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3301 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3361 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3421 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3481 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3541 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3601 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3661 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3721 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3781 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaacgaag catctgtgct
     3841 tcattttgta gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     3901 ctgcattttt acagaacaga aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg
     3961 tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat
     4021 ctgagctgca tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga
     4081 atctatactt cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa
     4141 gcatcttaga ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt
     4201 tttgcactgt aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc
     4261 ataaaaaaag cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca
     4321 ttttttcaag ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt
     4381 tgtgaacaga aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt
     4441 cttctatttt gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg
     4501 attcactcta tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata
     4561 aacataaaaa atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg
     4621 taggttatat agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt
     4681 tgtggaagcg gtattcgcaa tattttagta gctcgttaca gtccggtgcg tttttggttt
     4741 tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag ttcctatact
     4801 ttctagagaa taggaacttc ggaataggaa cttcaaagcg tttccgaaaa cgagcgcttc
     4861 cgaaaatgca acgcgagctg cgcacataca gctcactgtt cacgtcgcac ctatatctgc
     4921 gtgttgcctg tatatatata tacatgagaa gaacggcata gtgcgtgttt atgcttaaat
     4981 gcgtacttat atgcgtctat ttatgtagga tgaaaggtag tctagtacct cctgtgatat
     5041 tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta gctgttctat
     5101 atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt cctttgatat
     5161 tggatcatct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     5221 aggccctttc gtc
//
