LOCUS       sequence-332210-        5999 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 5999)
  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 5999)
  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-Pers
FEATURES             Location/Qualifiers
     source          1..5999
                     /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        859..1107
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1128..1203
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1243..1690
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1722..2429
                     /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(1867..1885)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2135..2154
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2746..3222
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2945..2964)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3262..3402
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3262..3391
                     /label=AAV2 ITR
     rep_origin      3477..3932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3564..3583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3774..3795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3949..3968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4068..4090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4128..4146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4214..4318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4319..4387,4388..5179)
                     /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(4537..4556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5350..5938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5839..5858
                     /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 accggatgcg ctcggcaatg agtgttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct tttttactag tgagggccta 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 ggatggcgag catcaagggc gttttagagc
      781 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      841 cggtgctttt ttgctagcga gggcctattt cccatgattc cttcatattt gcatatacga
      901 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
      961 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1021 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     1081 tatatcttgt ggaaaggacg aaacaccgag cacaacccct ccacgaggtt ttagagctag
     1141 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1201 tgctttttta ccggttatag actgcagagg gccctgcgta tgagtgcaag tgggttttag
     1261 gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa
     1321 gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag
     1381 gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc
     1441 gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg
     1501 tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc
     1561 cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg
     1621 cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct
     1681 gagagcgcag tcgagaaggt accggatcct ctagagtcga catggtgagc aagggcgagg
     1741 aggataacat ggccatcatc gaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     1801 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1861 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     1921 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1981 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     2041 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     2101 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     2161 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     2221 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     2281 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     2341 cctcccacga cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     2401 ccaccggcgg catggacgag ctgtacaagg aattctccgg actcagatct cgagaggagg
     2461 aggaggggac agacagcagg atgccccacc tcgacagccc cggcagctcc cagccgagac
     2521 gctccttcct ctcaagggtg atcagggcag cgctaccgtt gcagctgctt ctgctgctgc
     2581 tgctgctcct ggcctgcctg ctacctgcct ctgaagatga ctacagctgc acccaggcca
     2641 acaactttgc ccgatccttc taccccatgc tgcggtacac caacgggcca cctcccacct
     2701 aggatatcaa gcttatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     2761 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     2821 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     2881 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     2941 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     3001 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     3061 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     3121 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     3181 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     3241 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3301 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3361 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3421 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3481 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3541 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3601 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3661 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3721 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3781 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     3841 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3901 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3961 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4021 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4081 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4141 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4201 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4261 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4321 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4381 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4441 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4501 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4561 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4621 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4681 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4741 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4801 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4861 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4921 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4981 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5041 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5101 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5161 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5221 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5281 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5341 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5401 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5461 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5521 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5581 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5641 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5701 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5761 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5821 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5881 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5941 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
