LOCUS       sequence-60249-e        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of customizable guide RNA (gRNA) under cotnrol of murine 
            U6 promoter, also contains a CMV-puro-t2A-mCherry expression 
            cassette for mammalian gene knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 8308)
  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: 
            "10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28- 
            28"; volume: "152"; issue: "5"; pages: "1173-83"
COMMENT     Sequence Label: pgRNA-humanized
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(209..242)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     246..266
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        349..424
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        549..852
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        853..1056
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1090..1686
                     /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(1090..1109)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1586..1606
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1696..1749
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1756..1765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1762..2472
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1907..1925)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2175..2194
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2492..2525)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2581..3169
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2634..2654)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3052..3063)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3172..3188)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3173..3189)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3698..3878
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3940..4528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4020..4039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4699..5490,5491..5559))
                     /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     5322..5341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5560..5664)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5748..5767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5939..6318
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6320..6518
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6536..6716
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6763..6888
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7387..7620
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7805..7849
                     /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"
     misc_feature    8147..8264
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga
       61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag
      121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct
      181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt
      241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat
      301 aagtatccct tggagaacca ccttgttgga ccaggatggg caccacccgt tttagagcta
      361 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      421 gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg
      481 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      541 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      601 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      661 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      721 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      781 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      841 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      901 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      961 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1021 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     1081 tagcccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     1141 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     1201 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     1261 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     1321 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     1381 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     1441 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     1501 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     1561 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     1621 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     1681 ggtgccggat cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     1741 cctggcccac cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc
     1801 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1861 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1921 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     1981 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     2041 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     2101 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     2161 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     2221 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     2281 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     2341 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     2401 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     2461 ctgtacaagt aagaattcgt cgagggacct aataacttcg tatagcatac attatacgaa
     2521 gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat
     2581 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2641 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2701 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     2761 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     2821 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     2881 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2941 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3001 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3061 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3121 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     3181 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
     3241 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
     3301 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     3361 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     3421 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
     3481 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
     3541 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
     3601 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
     3661 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
     3721 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     3781 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     3841 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
     3901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4381 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4681 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4741 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4801 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4861 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4921 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4981 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5041 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5101 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5161 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5221 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5281 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5341 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5401 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5461 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5521 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5581 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5641 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcttaagc
     5701 ggtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     5761 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     5821 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     5881 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
     5941 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     6001 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     6061 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     6121 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     6181 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     6241 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     6301 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     6361 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     6421 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     6481 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
     6541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     6721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     6781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     6841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     6901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     6961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     7021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     7081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     7141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     7201 aaaagtaaga ccaccgcaca gcaagcggcc ggccgcgctg atcttcagac ctggaggagg
     7261 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     7321 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     7381 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     7441 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     7501 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     7561 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     7621 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     7681 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     7741 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     7801 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     7861 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     7921 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     7981 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     8041 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     8101 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     8161 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     8221 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     8281 acagcagaga tccagtttgg ttagtacc
//
