LOCUS       sequence-308671-        7625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA 
            targeting GFP and Luciferase using SpCas9 and LbCas12a nucleases 
            (CHyMErA system), respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7625)
  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 7625)
  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_GFP_Luciferase
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(20..25,26..43,44..50)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    58..74
                     /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     63..85
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     82..98
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     82..98
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     117..137
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        119..137
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        165..391
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             392..572
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    619..744
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1237..1470
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1655..1699
                     /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(2039..2287)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2097..2116)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2267..2287)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2329..2446
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2495..3005
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2924..2942
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3027..3626
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APCNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3027..3046)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3523..3543
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3642..4230
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3695..3715)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4113..4124)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4302..4535
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4613..4734
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4650..4669)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4704..4723
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4774..4909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4929..4948)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4930..4948)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4958..4975)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4958..4974)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4967..4989)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5116..5571
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5203..5222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5413..5434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5597..5701
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5702..5770,5771..6562)
                     /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(5920..5939)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6733..7321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7222..7241
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7475..7492
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    join(7609..7625,1..5)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
       61 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
      121 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
      181 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
      241 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
      301 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
      361 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
      421 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      481 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      541 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      601 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      661 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      721 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
      781 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
      841 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
      901 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
      961 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1021 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1081 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     1141 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     1201 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     1261 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     1321 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     1381 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     1441 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     1501 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     1561 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     1621 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     1681 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     1741 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     1801 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     1861 cccgagggga cccagagaat tcaaaaaaaa gggcgcaact gcaactccga atctacactt
     1921 agtagaaatt agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     1981 tcaacttgct atgctgtttc cagcatagct ctgaaacaca accactacct gagcacccgg
     2041 tacctcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
     2101 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
     2161 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
     2221 taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
     2281 ggccctcgaa ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2341 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2401 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2461 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggttgcgc cttttccaag
     2521 gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg
     2581 gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg
     2641 ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt
     2701 ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcagctcac actagtaccc
     2761 tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca
     2821 atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg
     2881 cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca
     2941 agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc
     3001 cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca
     3061 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3121 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3181 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3241 ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3301 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3361 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggtgtttcgc
     3421 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3481 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ctgcaacctc cccttctacg
     3541 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3601 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
     3661 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3721 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3781 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3841 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3901 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3961 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4021 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4081 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     4141 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4201 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     4261 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4321 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4381 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     4441 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     4501 cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct
     4561 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4621 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4681 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4741 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4801 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4861 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4921 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4981 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     5041 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5101 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5161 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5221 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5281 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5341 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5401 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5461 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5521 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5701 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5761 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5821 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5881 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5941 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6001 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6061 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6121 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6181 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6241 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6301 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6361 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6421 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6481 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6541 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6601 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6661 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6721 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6781 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6841 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6901 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6961 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7021 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7081 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7141 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7201 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7261 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7321 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7381 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7441 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7501 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7561 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7621 gctca
//
