LOCUS       sequence-308535-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA 
            targeting two intergenic sites in the human genome using SpCas9 and 
            LbCas12a nucleases (CHyMErA system), respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  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 7628)
  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: pLCHKO_intergenic-intergenic_1
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    4..25
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(40..45,46..63,64..70)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    78..94
                     /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     83..105
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     102..118
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     102..118
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     137..157
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        139..157
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        185..411
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             412..592
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    639..764
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1257..1490
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1675..1719
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        complement(2062..2310)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2120..2139)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2290..2310)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2352..2469
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2518..3028
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2947..2965
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3050..3649
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3050..3069)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3546..3566
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3665..4253
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3718..3738)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4136..4147)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4325..4558
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4636..4757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4673..4692)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4727..4746
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4797..4932
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4952..4971)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4953..4971)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4981..4998)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4981..4997)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4990..5012)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5139..5594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5226..5245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5436..5457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5620..5724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5725..5793,5794..6585)
                     /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     complement(5943..5962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6756..7344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7245..7264
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7498..7515
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
       61 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      121 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      181 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      241 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      301 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      361 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      421 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      481 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      541 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      601 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      661 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      721 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      781 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      841 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      901 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      961 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1021 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1081 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1141 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1201 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1261 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1321 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1381 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1441 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1501 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1561 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1621 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1681 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1741 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1801 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1861 tttcagaccc acctcccaac cccgagggga cccagagaat tcaaaaaaaa agaggattga
     1921 ctaactcaat tctatctaca cttagtagaa attagcaccg actcggtgcc actttttcaa
     1981 gttgataacg gactagcctt atttcaactt gctatgctgt ttccagcata gctctgaaac
     2041 caatcgctat ggaagagtca cggtacctcg tcctttccac aagatatata aagccaagaa
     2101 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     2161 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     2221 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     2281 atatgaagga atcatgggaa ataggccctc gaattctaga tcttgagaca aatggcagta
     2341 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     2401 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     2461 caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg gctcgagggg
     2521 gcccgggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg
     2581 gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt
     2641 tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc
     2701 tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga
     2761 agccgcagct cacactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg
     2821 ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg
     2881 ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc
     2941 cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt
     3001 tgaccgaatc accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta
     3061 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     3121 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     3181 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     3241 gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc
     3301 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3361 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3421 cctggccacc gtcggtgttt cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3481 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3541 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3601 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
     3661 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3721 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3781 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3841 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3901 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3961 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4021 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4081 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4141 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4201 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4261 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4321 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     4381 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4441 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4501 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     4561 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4621 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4681 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4741 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     4801 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4861 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4921 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4981 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5041 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5101 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5161 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5221 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5281 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5341 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5401 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5461 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5521 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5581 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5641 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5701 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5761 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5821 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5881 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5941 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6001 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6061 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6121 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6181 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6241 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6301 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6361 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6421 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6481 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6541 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6601 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6661 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6721 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6781 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6841 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6901 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6961 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7021 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7081 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7141 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7201 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7261 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7321 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7381 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7441 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7501 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7561 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7621 cgcaacgc
//
