LOCUS       sequence-270272-        5227 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 5227)
  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 5227)
  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..5227
                     /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        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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        1302..1570
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      1607..1626
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1664..1680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1664..1680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1677..1699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1688..1704
                     /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(1712..1718,1719..1736,1737..1742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1757..1778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1895..1912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2066..2654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2146..2165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2825..3616,3617..3685))
                     /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     3448..3467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3686..3790)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3817..5159
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4780..4827)
                     /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     5200..5218
                     /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 cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtcgt
     1081 ttctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctgtgctcga gtctttgaaa agataatgta
     1321 tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa
     1381 ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc
     1441 ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac
     1501 gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag
     1561 ataaatgatc aatttctact aagtgtagat ggagaccgag gtctcctttt tttgtttttt
     1621 atgtctgagc tccctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg
     1681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2941 gccccagtgc tgcaatgata ccgcggctcc cacgctcacc ggctccagat ttatcagcaa
     3001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3781 gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt
     3841 gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     3901 tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca
     3961 tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     4021 gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata
     4081 cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt
     4141 agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac
     4201 tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa
     4261 aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc
     4321 aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac
     4381 agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat
     4441 tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact
     4501 ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa
     4561 aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta
     4621 tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa
     4681 gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa
     4741 gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctaga
     4801 gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat
     4861 gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc
     4921 ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact
     4981 tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca
     5041 ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc
     5101 cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga tattggatca
     5161 tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     5221 tttcgtc
//
