LOCUS       sequence-308444-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of PDPR exon using SpCas9 and LbCas12a nucleases (CHyMErA 
            system).
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_PDPR_exon_deletion_4
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    483..716
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             901..945
                     /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(1288..1536)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1346..1365)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1516..1536)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1578..1695
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1744..2254
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2173..2191
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2276..2875
                     /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(2276..2295)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2772..2792
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2891..3479
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2944..2964)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3362..3373)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3551..3784
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3862..3983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3899..3918)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3953..3972
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4023..4158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4178..4197)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4179..4197)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4207..4224)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4207..4223)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4216..4238)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4365..4820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4452..4471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4662..4683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4846..4950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4951..5019,5020..5811)
                     /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(5169..5188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5982..6570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6471..6490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6724..6741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6858..6879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6894..6899,6900..6917,6918..6924)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6932..6948
                     /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     6937..6959
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6956..6972
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6956..6972
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6991..7011
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6993..7011
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7039..7265
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7266..7446
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7493..7618
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
       61 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
      121 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
      181 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
      241 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
      301 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
      361 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
      421 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
      481 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
      541 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
      601 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
      661 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
      721 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
      781 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
      841 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
      901 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
      961 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1021 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     1081 ttatcgtttc agacccacct cccaaccccg aggggaccca gagaattcaa aaaaaaacat
     1141 ggacaagaga ggaggcgtaa tctacactta gtagaaatta gcaccgactc ggtgccactt
     1201 tttcaagttg ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc
     1261 tgaaacgtca catcagttct tcttcccggt acctcgtcct ttccacaaga tatataaagc
     1321 caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa
     1381 ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa
     1441 tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat
     1501 atgcaaatat gaaggaatca tgggaaatag gccctcgaat tctagatctt gagacaaatg
     1561 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     1621 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     1681 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     1741 gagggggccc gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg
     1801 ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac
     1861 gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct
     1921 tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca
     1981 aacggaagcc gcagctcaca ctagtaccct cgcagacgga cagcgccagg gagcaatggc
     2041 agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag
     2101 cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt
     2161 cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc
     2221 cctcgttgac cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac
     2281 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2341 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     2401 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2461 caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt
     2521 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     2581 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     2641 gtggttcctg gccaccgtcg gtgtttcgcc cgaccaccag ggcaagggtc tgggcagcgc
     2701 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     2761 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     2821 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     2881 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2941 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3001 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3061 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3121 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3181 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3241 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3301 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3361 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3421 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     3481 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     3541 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     3601 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     3661 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     3721 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     3781 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     3841 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     3901 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3961 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     4021 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4081 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4141 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     4201 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4261 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4321 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     4381 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4441 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4501 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4561 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4621 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4681 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4741 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4801 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4861 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4921 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4981 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5041 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5101 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5161 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5221 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5281 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5341 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5401 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5461 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5521 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5581 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5641 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5701 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5761 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5821 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5881 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5941 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6001 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6061 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6121 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6181 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6241 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6301 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6361 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6421 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6481 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6541 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6601 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6661 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6721 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     6781 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6841 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6901 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     6961 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     7021 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
     7081 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
     7141 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
     7201 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
     7261 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     7321 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     7381 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     7441 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
     7501 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     7561 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     7621 tattaagc
//
