LOCUS       sequence-315172-        3640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO vector for the cloning of _the SpCas9 tracrRNA - AsCas12a 
            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 3640)
  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 3640)
  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_AsCas12a.DR
FEATURES             Location/Qualifiers
     source          1..3640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     355..374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     608..625
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    742..763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(778..783,784..801,802..808)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    816..832
                     /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     821..843
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     840..856
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     840..856
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        874..892
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     874..891
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1163..1182)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1164..1182)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1189..1206)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1189..1205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1198..1220)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1343..1645
                     /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     1524..1543
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1994..2788
                     /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             1994..2788
                     /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(2048..2067)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2658..2677
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2988..2997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2994..3368
                     /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      join(3506..3640,1..454)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
       61 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      481 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      541 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      601 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
      661 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      721 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      781 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      841 aggaaacagc tatgaccatg attacgccaa gctatttagg tgacactata gaatactcaa
      901 gctatgcatc aagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg
      961 aattcgccct taacgtctct atctacaaga gtagaaatta gcaccgactc ggtgccactt
     1021 tttcaagttg ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc
     1081 tgaaacagag acgaagggcg aattctgcag atatccatca cactggcggc cgctcgagca
     1141 tgcatctaga gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
     1201 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     1261 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1321 gttgcgcagc ctatacgtac ggcagtttaa ggtttacacc tataaaagag agagccgtta
     1381 tcgtctgttt gtggatgtac agagtgatat tattgacacg ccggggcgac ggatggtgat
     1441 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     1501 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     1561 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     1621 taacctgatg ttctggggaa tataaatgtc aggcatgaga ttatcaaaaa ggatcttcac
     1681 ctagatcctt ttcacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt
     1741 cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg
     1801 cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga
     1861 attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
     1921 tttcttgccg ccaaggatct gatggcgcag gggatcaagc tctgatcaag agacaggatg
     1981 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     2041 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     2101 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     2161 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     2221 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     2281 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     2341 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     2401 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     2461 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     2521 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     2581 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     2641 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     2701 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     2761 ctatcgcctt cttgacgagt tcttctgaat tattaacgct tacaatttcc tgatgcggta
     2821 ttttctcctt acgcatctgt gcggtatttc acaccgcatc aggtggcact tttcggggaa
     2881 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2941 tgagacaata accctgataa atgcttcaat aatagcacgt gaggagggcc accatggcca
     3001 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     3061 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     3121 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     3181 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     3241 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     3301 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     3361 aggactgaca cgtgctaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3421 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3481 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3541 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3601 ctctttttcc gaaggtaact ggcttcagca gagcgcagat
//
