LOCUS       sequence-308590-        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_2
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    391..624
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             809..853
                     /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(1196..1444)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1254..1273)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1424..1444)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1486..1603
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1652..2162
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2081..2099
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2184..2783
                     /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(2184..2203)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2680..2700
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2799..3387
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2852..2872)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3270..3281)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3459..3692
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3770..3891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3807..3826)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3861..3880
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3931..4066
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4086..4105)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4087..4105)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4115..4132)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4115..4131)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4124..4146)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4273..4728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4360..4379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4570..4591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4754..4858
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4859..4927,4928..5719)
                     /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(5077..5096)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5890..6478
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6379..6398
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6632..6649
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6766..6787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6802..6807,6808..6825,6826..6832)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6840..6856
                     /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     6845..6867
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6864..6880
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6864..6880
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6899..6919
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6901..6919
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6947..7173
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7174..7354
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7401..7526
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
       61 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
      121 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
      181 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
      241 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
      301 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
      361 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
      421 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
      481 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
      541 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
      601 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
      661 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
      721 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
      781 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
      841 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
      901 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
      961 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     1021 gggacccaga gaattccaaa aaaaaattaa ctcagaatag cttcccaatc tacacttagt
     1081 agaaattagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttca
     1141 acttgctatg ctgtttccag catagctctg aaacctcttg tccatgttaa tagtcggtac
     1201 ctcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     1261 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     1321 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa
     1381 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     1441 cctcgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1501 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1561 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1621 cagcagagat ccactttggc gccggctcga gggggcccgg gttgcgcctt ttccaaggca
     1681 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     1741 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     1801 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     1861 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc agctcacact agtaccctcg
     1921 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     1981 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     2041 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     2101 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     2161 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2221 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2281 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     2341 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg
     2401 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     2461 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     2521 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggt gtttcgcccg
     2581 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     2641 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     2701 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     2761 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     2821 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     2881 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     2941 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3001 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3061 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3121 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3181 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3241 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3301 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3361 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     3421 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     3481 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     3541 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     3601 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     3661 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     3721 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     3781 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3841 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3901 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3961 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4021 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     4081 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     4141 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4201 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4261 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4321 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4381 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4441 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4501 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4561 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4621 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4681 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4801 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4861 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4921 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4981 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5041 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5101 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5161 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5221 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5281 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5341 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5401 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5461 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5521 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5581 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5641 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5701 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5761 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5821 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5881 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5941 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6001 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6061 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6121 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6181 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6241 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6301 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6361 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6421 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6481 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6541 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6601 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6661 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     6721 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6781 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6841 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     6901 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
     6961 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
     7021 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     7081 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     7141 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     7201 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     7261 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     7321 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     7381 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     7441 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     7501 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     7561 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     7621 caagcagg
//
