LOCUS       sequence-389127-        5688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA(CRY2 gene) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5688)
  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 5688)
  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: sgCRY2
FEATURES             Location/Qualifiers
     source          1..5688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(299..318)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     418..440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(478..496)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        564..668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(669..737,738..1529)
                     /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(887..906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1700..2288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2189..2208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2350..2479
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2510..2758
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2780..2855
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2880..3128
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3150..3225
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3281..3728
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             3760..4467
                     /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(3905..3923)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4173..4192
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4784..5260
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4983..5002)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5300..5440
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5300..5429
                     /label=AAV2 ITR
     rep_origin      join(5515..5688,1..282)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5602..5621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
       61 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      121 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
      181 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
      241 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
      301 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
      361 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      421 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
      481 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
      541 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      601 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      661 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      721 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      781 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      841 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      901 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      961 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1021 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1081 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     1141 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     1201 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     1261 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     1321 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     1381 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     1441 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     1501 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1561 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1621 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1681 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1741 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1801 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1861 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1921 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1981 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2041 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2101 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2161 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2221 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2281 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2341 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     2401 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     2461 tccatcacta ggggttcctg cggccgcacg cgtcacctgc taacaattgg agggcctatt
     2521 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2581 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2641 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2701 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc
     2761 tgtgggcatc aaccgatggg ttttagagct agaaatagca agttaaaata aggctagtcc
     2821 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgctagcgct agcgctagcg
     2881 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2941 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     3001 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     3061 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     3121 gaaacaccgt cggttgatgc ccacagacgg ttttagagct agaaatagca agttaaaata
     3181 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tcttaagtta
     3241 gcaggtgacc ggttatagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
     3301 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
     3361 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
     3421 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
     3481 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
     3541 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
     3601 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
     3661 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
     3721 gagcgcagtc gagaaggtac cggatcctct agagtcgaca tggtgagcaa gggcgaggag
     3781 gataacatgg ccatcatcga ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     3841 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     3901 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     3961 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     4021 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     4081 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     4141 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     4201 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     4261 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     4321 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     4381 tcccacgacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     4441 accggcggca tggacgagct gtacaaggaa ttctccggac tcagatctcg agaggaggag
     4501 gaggggacag acagcaggat gccccacctc gacagccccg gcagctccca gccgagacgc
     4561 tccttcctct caagggtgat cagggcagcg ctaccgttgc agctgcttct gctgctgctg
     4621 ctgctcctgg cctgcctgct acctgcctct gaagatgact acagctgcac ccaggccaac
     4681 aactttgccc gatccttcta ccccatgctg cggtacacca acgggccacc tcccacctag
     4741 gatatcaagc ttatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga
     4801 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     4861 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     4921 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     4981 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     5041 ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg
     5101 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     5161 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     5221 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac
     5281 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     5341 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     5401 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     5461 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5581 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttcc
//
