LOCUS       sequence-263332-        8284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of AsCas12a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8284)
  AUTHORS   DeWeirdt PC, Sanson KR, Sangree AK, Hegde M, Hanna RE, Feeley MN, 
            Griffith AL, Teng T, Borys SM, Strand C, Joung JK, Kleinstiver BP, 
            Pan X, Huang A, Doench JG
  TITLE     Optimization of AsCas12a for combinatorial genetic screens in human 
            cells.
  JOURNAL   Nat Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6.
  PUBMED    32661438
REFERENCE   2  (bases 1 to 8284)
  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"; journalName: "Nat 
            Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6."
COMMENT     Sequence Label: pRDA_052
FEATURES             Location/Qualifiers
     source          1..8284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    83..104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(119..124,125..142,143..149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    157..173
                     /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     162..184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     181..197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     181..197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     216..236
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        218..236
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        264..490
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             491..671
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    718..843
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1336..1569
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1754..1798
                     /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        1982..2230
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1982..2002
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2153..2172
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2338..2455
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2594..3772
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2825..3763
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3720..3740
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3785..4384
                     /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(3785..3804)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4281..4301
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4400..4988
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4453..4473)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4871..4882)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5060..5293
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5371..5492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5408..5427)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5462..5481
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5532..5667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5687..5706)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5688..5706)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5716..5733)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5716..5732)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5725..5747)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5874..6329
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5961..5980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6171..6192
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6355..6459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6460..6528,6529..7320)
                     /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(6678..6697)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7491..8079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7980..7999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8233..8250
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
       61 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      181 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      241 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
      301 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
      361 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
      421 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
      481 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      541 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      601 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      661 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      721 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      781 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      841 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      901 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
      961 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     1021 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     1081 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     1141 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1201 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1261 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1321 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1381 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1441 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1501 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1561 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1621 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1681 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1741 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     1801 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     1861 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     1921 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagaggtac
     1981 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2041 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2101 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2161 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2221 acgaaacacc ggtaatttct actcttgtag atggagacgc gttttttctc gtctctgaat
     2281 tcgctagcaa gcttggcgta actagatctt gagacaaatg gcagtattca tccacaattt
     2341 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2401 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2461 ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc gggtgcaaag
     2521 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     2581 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2641 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     2701 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2761 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2821 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
     2881 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     2941 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
     3001 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     3061 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     3121 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     3181 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     3241 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     3301 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     3361 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     3421 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     3481 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     3541 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     3601 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     3661 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     3721 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgtacggc
     3781 caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     3841 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     3901 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     3961 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcc gtggcggtct ggaccacgcc
     4021 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     4081 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     4141 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     4201 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     4261 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     4321 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     4381 ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     4441 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     4501 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     4561 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     4621 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     4681 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4741 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     4801 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4861 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4921 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4981 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5041 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     5101 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5161 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5221 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5281 aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt
     5341 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     5401 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5461 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     5521 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5581 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     5641 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     5701 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     5761 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     5821 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     5881 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5941 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6001 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6061 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     6121 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6181 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     6241 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6301 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     6361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6421 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6481 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6541 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6601 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6661 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6721 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6781 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6841 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6901 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6961 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7021 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7081 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7141 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     7201 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7261 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7321 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7381 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7621 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     8161 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     8221 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     8281 cctc
//
