LOCUS       sequence-300004-        3642 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 3642)
  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 3642)
  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..3642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(529..548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(530..548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(555..572)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(555..571)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(564..586)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             709..1011
                     /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     890..909
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1360..2154
                     /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             1360..2154
                     /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(1414..1433)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2024..2043
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2355..2364
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2361..2735
                     /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      2873..3461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3362..3381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3615..3632
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttaacg tctctatcta cacttagtag
      361 aaattagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttatttcaac
      421 ttgctatgct gtttccagca tagctctgaa acagagacga agggcgaatt ctgcagatat
      481 ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag
      541 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
      601 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
      661 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt
      721 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
      781 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
      841 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
      901 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
      961 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     1021 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag
     1081 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     1141 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     1201 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     1261 aagccctgca aagtaaactg gatggctttc tcgccgccaa ggatctgatg gcgcagggga
     1321 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     1381 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     1441 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     1501 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     1561 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     1621 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     1681 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     1741 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     1801 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     1861 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     1921 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     1981 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2041 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2101 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt
     2161 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2221 cgcatacagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2281 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2341 agcacgtgag gagggccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     2401 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     2461 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     2521 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     2581 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     2641 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     2701 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctaaaactt catttttaat
     2761 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2821 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2881 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2941 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3001 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3061 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3121 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3181 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3241 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3301 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3361 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3421 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3481 ttttacggtt cctgggcttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3541 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3601 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag
//
