LOCUS       sequence-190219-        8297 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 8297)
  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 8297)
  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..8297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(205..238)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     242..262
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        345..420
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        545..848
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        849..1052
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1086..1682
                     /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(1086..1105)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1582..1602
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1692..1745
                     /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      1752..1761
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1758..2468
                     /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(1903..1921)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2171..2190
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2488..2521)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2577..3165
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2630..2650)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3048..3059)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3168..3184)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3169..3185)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3694..3874
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3936..4524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4016..4035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4695..5486,5487..5555))
                     /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     5318..5337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5556..5660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5735..5754)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5926..6305
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6307..6505
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6523..6703
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6750..6875
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7372..7605
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7790..7834
                     /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    8132..8249
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ccgctctaga gatccgacgc gccatctcta ggcccgcgcc ggccccctcg cacggacttg
       61 tgggagaagc tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac
      121 tatagaggct taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat
      181 tttacatgat aggcttggat ttctataact tcgtatagca tacattatac gaagttataa
      241 acagcacaaa aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataagt
      301 atcccttgga gaaccacctt gttggaccag gatgggcacc acccgtttta gagctagaaa
      361 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      421 tttttttctc gagtactagg atccattagg cggccgcgtg gataaccgta ttaccgccat
      481 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      541 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      601 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      661 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      721 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      781 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      841 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      901 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      961 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1021 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc
     1081 ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg
     1141 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc
     1201 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     1261 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     1321 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     1381 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     1441 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     1501 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     1561 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     1621 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     1681 ccggatcggg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg
     1741 gcccaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     1801 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     1861 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1921 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1981 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     2041 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     2101 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     2161 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     2221 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     2281 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     2341 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     2401 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     2461 acaagtaaga attcgtcgag ggacctaata acttcgtata gcatacatta tacgaagtta
     2521 tacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc
     2581 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2641 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     2701 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     2761 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     2821 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     2881 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     2941 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3001 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3061 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3121 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac
     3181 ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat
     3241 gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct
     3301 caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     3361 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg
     3421 tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg
     3481 atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag
     3541 gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg
     3601 atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat
     3661 cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga
     3721 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     3781 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     3841 agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag
     3901 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3961 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4021 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4081 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4141 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4201 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4261 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4321 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4381 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4441 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4501 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4561 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4621 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4681 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4741 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4801 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4861 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4921 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4981 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5041 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5101 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5161 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5221 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5281 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5341 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5401 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5461 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5521 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5581 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5641 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     5701 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5761 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     5821 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     5881 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     5941 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     6001 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     6061 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     6121 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     6181 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     6241 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     6301 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     6361 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     6421 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     6481 tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac
     6541 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     6601 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     6661 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
     6721 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
     6781 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
     6841 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
     6901 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
     6961 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     7021 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     7081 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     7141 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     7201 ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     7261 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     7321 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     7381 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     7441 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     7501 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     7561 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     7621 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     7681 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     7741 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     7801 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     7861 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     7921 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     7981 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     8041 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
     8101 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     8161 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     8221 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     8281 tttggttagt accgggc
//
