LOCUS       sequence-308438-        7627 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of HPRT1 exon 2 using SpCas9 and AsCas12a nucleases 
            (CHyMErA system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  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 7627)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_HPRT1_exon2_1_As
FEATURES             Location/Qualifiers
     source          1..7627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    592..825
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1010..1054
                     /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(1396..1644)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1454..1473)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1624..1644)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1686..1803
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1852..2362
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2281..2299
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2384..2983
                     /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(2384..2403)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2880..2900
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2999..3587
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3052..3072)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3470..3481)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3659..3892
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3970..4091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4007..4026)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4061..4080
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4131..4266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4286..4305)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4287..4305)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4315..4332)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4315..4331)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4324..4346)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4473..4928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4560..4579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4770..4791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4954..5058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5059..5127,5128..5919)
                     /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(5277..5296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6090..6678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6579..6598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6832..6849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6966..6987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7002..7007,7008..7025,7026..7032)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7040..7056
                     /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     7045..7067
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7064..7080
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7064..7080
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7099..7119
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7101..7119
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7147..7373
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7374..7554
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    join(7601..7627,1..99)
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
       61 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
      121 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
      181 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
      241 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
      301 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
      361 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
      421 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
      481 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
      541 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
      601 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
      661 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
      721 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
      781 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
      841 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
      901 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
      961 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     1021 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     1081 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     1141 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     1201 gacccacctc ccaaccccga ggggacccag agaattcaaa aaaaaaggcc ctcctctttt
     1261 atttttaaat ctacaagagt agaaattagc accgactcgg tgccactttt tcaagttgat
     1321 aacggactag ccttatttca acttgctatg ctgtttccag catagctctg aaactgtaac
     1381 tatatgtctt agtccggtac ctcgtccttt ccacaagata tataaagcca agaaatcgaa
     1441 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc
     1501 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt
     1561 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga
     1621 aggaatcatg ggaaataggc cctcgaattc tagatcttga gacaaatggc agtattcatc
     1681 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     1741 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     1801 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gggggcccgg
     1861 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
     1921 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag
     1981 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
     2041 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
     2101 agctcacact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
     2161 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
     2221 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     2281 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     2341 aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc
     2401 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2461 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     2521 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2581 cgcggacgac ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     2641 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     2701 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     2761 caccgtcggt gtttcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     2821 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     2881 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     2941 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     3001 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3061 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3121 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3181 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3241 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3301 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3361 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3421 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3481 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3541 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     3601 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     3661 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     3721 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     3781 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     3841 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     3901 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     3961 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4021 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4081 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4141 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4201 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4261 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     4321 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4381 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4441 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     4501 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4561 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4621 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4681 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4741 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4801 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4861 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4921 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4981 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5041 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5101 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5161 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5221 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5281 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5341 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5401 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5461 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5521 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5581 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5641 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5701 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5761 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5821 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5881 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5941 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6001 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6061 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6361 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6661 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6721 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6781 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6841 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     6901 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     6961 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     7021 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     7081 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     7141 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
     7201 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
     7261 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
     7321 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
     7381 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     7441 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     7501 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     7561 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     7621 gcttgct
//
