LOCUS       sequence-325988-        6001 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 6001)
  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 6001)
  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-Crys
FEATURES             Location/Qualifiers
     source          1..6001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        178..253
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        266..514
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        536..611
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        651..1098
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1130..1837
                     /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(1275..1293)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1543..1562
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2154..2630
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2353..2372)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2670..2810
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2670..2799
                     /label=AAV2 ITR
     rep_origin      2885..3340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2972..2991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3182..3203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3357..3376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3476..3498
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3536..3554)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3622..3726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3727..3795,3796..4587)
                     /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(3945..3964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4758..5346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5247..5266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5408..5537
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5552..5800
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5821..5896
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        join(5909..6001,1..156)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
       61 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      121 ttggctttat atatcttgtg gaaaggacga aacaccgctg tgggcatcaa ccgatgggtt
      181 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      241 accgagtcgg tgcttttttg ctagcgaggg cctatttccc atgattcctt catatttgca
      301 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
      361 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      421 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      481 ggctttatat atcttgtgga aaggacgaaa caccgtcggt tgatgcccac agacggtttt
      541 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      601 cgagtcggtg cttttttacc ggttatagac tgcagagggc cctgcgtatg agtgcaagtg
      661 ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca
      721 ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg
      781 ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct
      841 tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca
      901 ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc
      961 ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg
     1021 cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg
     1081 tcgtgcctga gagcgcagtc gagaaggtac cggatcctct agagtcgaca tggtgagcaa
     1141 gggcgaggag gataacatgg ccatcatcga ggagttcatg cgcttcaagg tgcacatgga
     1201 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     1261 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     1321 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     1381 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     1441 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     1501 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     1561 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     1621 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     1681 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     1741 ggacatcacc tcccacgacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     1801 ccgccactcc accggcggca tggacgagct gtacaaggaa ttctccggac tcagatctcg
     1861 agaggaggag gaggggacag acagcaggat gccccacctc gacagccccg gcagctccca
     1921 gccgagacgc tccttcctct caagggtgat cagggcagcg ctaccgttgc agctgcttct
     1981 gctgctgctg ctgctcctgg cctgcctgct acctgcctct gaagatgact acagctgcac
     2041 ccaggccaac aactttgccc gatccttcta ccccatgctg cggtacacca acgggccacc
     2101 tcccacctag gatatcaagc ttatcgatac cgagcgctgc tcgagagatc tacgggtggc
     2161 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     2221 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     2281 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     2341 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     2401 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     2461 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     2521 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     2581 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt
     2641 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     2701 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     2761 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     2821 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     2881 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2941 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     3001 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3061 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     3121 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3181 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     3241 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     3301 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     3361 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     3421 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3481 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     3541 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3721 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3781 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3841 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3901 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3961 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4021 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4081 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4141 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4201 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4261 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4321 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4381 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4441 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4501 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4561 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4621 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4681 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4741 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4801 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4861 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4921 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4981 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5041 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5101 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5161 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5221 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5281 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5341 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5401 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
     5461 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
     5521 catcactagg ggttcctgcg gccgcacgcg tgagggccta tttcccatga ttccttcata
     5581 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     5641 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     5701 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     5761 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtcgaggat atagacgcag
     5821 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     5881 ggcaccgagt cggtgctttt ttacatgtga gggcctattt cccatgattc cttcatattt
     5941 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     6001 g
//
