LOCUS       sequence-348670-        5279 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 5279)
  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 5279)
  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: sgCRY1
FEATURES             Location/Qualifiers
     source          1..5279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        795..1043
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     795..815
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     966..985
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1064..1139
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1173..1620
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1652..2359
                     /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(1797..1815)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2065..2084
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2676..3152
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2875..2894)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3192..3332
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3192..3321
                     /label=AAV2 ITR
     rep_origin      3407..3862
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3494..3513)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3704..3725
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3879..3898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3998..4020
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4058..4076)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4144..4248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4249..4317,4318..5109)
                     /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(4467..4486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat atacgataca
      841 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      901 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      961 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1021 tcttgtggaa aggacgaaac accggtcgag gatatagacg caggttttag agctagaaat
     1081 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1141 ttttttctag actgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg
     1201 aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc
     1261 cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga
     1321 ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc
     1381 gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa
     1441 actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac
     1501 cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac
     1561 tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag
     1621 tcgagaaggt accggatcct ctagagtcga catggtgagc aagggcgagg aggataacat
     1681 ggccatcatc gaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1741 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1801 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1861 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1921 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1981 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     2041 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     2101 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     2161 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     2221 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacga
     2281 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2341 catggacgag ctgtacaagg aattctccgg actcagatct cgagaggagg aggaggggac
     2401 agacagcagg atgccccacc tcgacagccc cggcagctcc cagccgagac gctccttcct
     2461 ctcaagggtg atcagggcag cgctaccgtt gcagctgctt ctgctgctgc tgctgctcct
     2521 ggcctgcctg ctacctgcct ctgaagatga ctacagctgc acccaggcca acaactttgc
     2581 ccgatccttc taccccatgc tgcggtacac caacgggcca cctcccacct aggatatcaa
     2641 gcttatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc
     2701 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     2761 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     2821 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     2881 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     2941 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     3001 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     3061 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     3121 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac
     3181 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3241 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3301 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     3361 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3421 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3481 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3541 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3601 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     3661 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3721 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     3781 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3841 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     3901 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3961 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4021 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4081 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4141 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4201 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4261 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4321 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4381 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4441 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4501 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4561 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4621 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4681 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4741 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4801 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4861 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4921 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4981 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5041 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5101 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5161 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5221 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
