LOCUS       sequence-332215-        5642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5642)
  AUTHORS   Kim B, Kim J, Chun M, Park I, Kwak D, Choi M, Kim K, Choe HK
  TITLE     Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to
            simultaneously knock out redundant clock genes.
  JOURNAL   Sci Rep. 2021 Jan 28;11(1):2575. doi: 10.1038/s41598-021-82287-0.
  PUBMED    33510438
REFERENCE   2  (bases 1 to 5642)
  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: "Sci Rep."; 
            date: "2021-01-28"; volume: "11(1):2575. doi"; pages: " 
            10.1038/s41598-021-82287-0"
COMMENT     Sequence Label: CSAC-Bmal1
FEATURES             Location/Qualifiers
     source          1..5642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        502..750
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        771..846
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        886..1333
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1365..2072
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIEEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHDEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1510..1528)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1778..1797
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2389..2865
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2588..2607)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2905..3045
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2905..3034
                     /label=AAV2 ITR
     rep_origin      3120..3575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3207..3226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3417..3438
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3592..3611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3711..3733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3771..3789)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3857..3961
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3962..4030,4031..4822)
                     /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(4180..4199)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4993..5581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5482..5501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaaccg gagagtaggt cgggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct tttttgctag cgagggccta tttcccatga ttccttcata tttgcatata
      541 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
      601 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      661 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      721 ttatatatct tgtggaaagg acgaaacacc gcctcttttc aatctgactg gttttagagc
      781 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      841 cggtgctttt ttaccggtta tagactgcag agggccctgc gtatgagtgc aagtgggttt
      901 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      961 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
     1021 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
     1081 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
     1141 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
     1201 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
     1261 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
     1321 cctgagagcg cagtcgagaa ggtaccggat cctctagagt cgacatggtg agcaagggcg
     1381 aggaggataa catggccatc atcgaggagt tcatgcgctt caaggtgcac atggagggct
     1441 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1501 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1561 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1621 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1681 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1741 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1801 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1861 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1921 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1981 tcacctccca cgacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2041 actccaccgg cggcatggac gagctgtaca aggaattctc cggactcaga tctcgagagg
     2101 aggaggaggg gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga
     2161 gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc
     2221 tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg
     2281 ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca
     2341 cctaggatat caagcttatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     2401 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     2461 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     2521 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     2581 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     2641 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     2701 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     2761 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     2821 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     2881 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     2941 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3001 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3061 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     3121 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3181 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3241 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3301 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     3361 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3421 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     3481 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3541 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     3601 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3661 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3721 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     3781 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3841 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3901 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3961 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4021 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4081 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4141 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4201 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4261 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4321 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4381 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4441 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4501 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4561 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4621 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4681 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4741 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4801 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4861 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4921 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4981 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5041 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5101 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5161 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5221 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5281 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5341 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5401 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5461 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5521 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5581 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5641 gt
//
