LOCUS       sequence-308669-        3641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO vector for the cloning of _the SpCas9 tracrRNA - LbCas12a 
            Direct Repeat (DR) fragment into the pLCHKO hgRNA vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3641)
  AUTHORS   Gonatopoulos-Pournatzis T, Aregger M, Brown KR, Farhangmehr S, 
            Braunschweig U, Ward HN, Ha KCH, Weiss A, Billmann M, Durbic T, 
            Myers CL, Blencowe BJ, Moffat J
  TITLE     Genetic interaction mapping and exon-resolution functional genomics 
            with a hybrid Cas9-Cas12a platform.
  JOURNAL   Nat Biotechnol. 2020 May;38(5):638-648. doi: 
            10.1038/s41587-020-0437-z. Epub 2020 Mar 16.
  PUBMED    32249828
REFERENCE   2  (bases 1 to 3641)
  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: 
            "10.1038/s41587-020-0437-z"; journalName: "Nat Biotechnol"; date: 
            "2020-05"; volume: "38"; issue: "5"; pages: "638-648"
COMMENT     Sequence Label: Topo_SpCas9.tracr_LbCas12a.DR
FEATURES             Location/Qualifiers
     source          1..3641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(23..41)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(48..65)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(48..64)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(57..79)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             202..504
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     383..402
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             853..1647
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             853..1647
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(907..926)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1517..1536
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      1847..1856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1853..2227
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      2365..2953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2854..2873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3107..3124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3241..3262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3277..3282,3283..3300,3301..3307)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3315..3331
                     /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     3320..3342
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3339..3355
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3339..3355
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3373..3391
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3373..3390
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
       61 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      121 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      181 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
      241 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
      301 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
      361 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
      421 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
      481 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
      541 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
      601 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
      661 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
      721 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
      781 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
      841 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
      901 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
      961 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1021 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1081 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1141 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1201 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1261 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1321 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1381 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1441 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1501 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     1561 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     1621 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     1681 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     1741 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1801 gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca ccatggccaa
     1861 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
     1921 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
     1981 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
     2041 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
     2101 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
     2161 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     2221 ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2281 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3001 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3061 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3121 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3181 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3241 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     3301 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     3361 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac
     3421 cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccctt aacgtctcta
     3481 tctacactta gtagaaatta gcaccgactc ggtgccactt tttcaagttg ataacggact
     3541 agccttattt caacttgcta tgctgtttcc agcatagctc tgaaacagag acgaagggcg
     3601 aattctgcag atatccatca cactggcggc cgctcgagca t
//
