LOCUS       sequence-164464-        8330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cpf1 customizable sgRNA from U6 promoter and puromycin 
            resistance from EF-1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8330)
  AUTHORS   Kim HK, Song M, Lee J, Menon AV, Jung S, Kang YM, Choi JW, Woo E, 
            Koh HC, Nam JW, Kim H
  TITLE     In vivo high-throughput profiling of CRISPR-Cpf1 activity.
  JOURNAL   Nat Methods. 2016 Dec 19. doi: 10.1038/nmeth.4104.
  PUBMED    27992409
REFERENCE   2  (bases 1 to 8330)
  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 
            Methods. 2016 Dec 19. doi: 10.1038/nmeth.4104."
COMMENT     Sequence Label: Lenti_gRNA-Puro
FEATURES             Location/Qualifiers
     source          1..8330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        86..1264
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          317..1255
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1212..1232
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1277..1876
                     /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(1277..1296)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1773..1793
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1892..2480
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1945..1965)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2363..2374)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2552..2785
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2863..2984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2900..2919)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2954..2973
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3024..3159
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3179..3198)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3180..3198)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3208..3225)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3208..3224)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3217..3239)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3366..3821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3453..3472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3663..3684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3847..3951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3952..4020,4021..4812)
                     /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(4170..4189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4983..5571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5472..5491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5725..5742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5859..5880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5895..5900,5901..5918,5919..5925)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5933..5949
                     /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     5938..5960
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5957..5973
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5957..5973
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5992..6012
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5994..6012
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6040..6266
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6267..6447
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6494..6619
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7112..7345
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7530..7574
                     /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        7752..8000
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7752..7772
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7923..7942
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    8160..8277
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ccgggtgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
       61 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
      121 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag
      181 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
      241 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
      301 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
      361 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
      421 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
      481 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
      541 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
      601 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
      661 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
      721 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
      781 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
      841 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
      901 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
      961 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1021 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1081 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     1141 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     1201 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     1261 gtgacgtacg gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     1321 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     1381 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     1441 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt
     1501 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     1561 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     1621 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     1681 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     1741 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     1801 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     1861 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     1921 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     1981 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     2041 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     2101 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     2161 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     2221 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     2281 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     2341 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     2401 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     2461 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     2521 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     2581 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     2641 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     2701 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     2761 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     2821 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     2881 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2941 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     3001 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3061 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3121 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     3181 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     3241 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     3301 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     3361 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3421 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3481 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3541 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3601 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3661 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3721 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3781 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3841 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3901 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3961 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4021 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4081 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4141 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4201 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4261 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4321 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4381 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4441 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4501 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4561 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4621 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4681 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4741 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4801 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4861 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4921 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4981 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5041 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5101 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5161 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5221 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5281 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5341 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5401 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5461 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5521 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5581 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5641 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5701 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5761 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5821 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5881 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5941 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     6001 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
     6061 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
     6121 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
     6181 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
     6241 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
     6301 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     6361 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     6421 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     6481 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     6541 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     6601 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     6661 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     6721 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     6781 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     6841 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     6901 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     6961 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     7021 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     7081 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     7141 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     7201 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     7261 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     7321 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     7381 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     7441 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     7501 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     7561 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     7621 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     7681 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     7741 ggggacccag agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     7801 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     7861 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     7921 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     7981 tgtggaaagg acgaaacacc ggagacgttg actatcgtct cgctactcta ccacttgtac
     8041 ttcagcggtc agcttactcg acttaacgtg cacgtgacac gttctagacc gtacatgctt
     8101 acatgggatg aagcttggcg taactagatc ttgagacaaa tggcagtatt catccacaat
     8161 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     8221 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     8281 gtttattaca gggacagcag agatccactt tggcgccggc tcgagggggc
//
