LOCUS       sequence-326387-        5653 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses sgRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5653)
  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 5653)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   249..389
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   260..389
                     /label=AAV2 ITR
     promoter        404..652
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        673..748
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        761..1009
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1030..1105
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1145..1592
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1624..2331
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1769..1787)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2037..2056
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2648..3124
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2847..2866)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3164..3304
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3164..3293
                     /label=AAV2 ITR
     rep_origin      3379..3834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3466..3485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3676..3697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3851..3870)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3970..3992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4030..4048)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4116..4220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4221..4289,4290..5081)
                     /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(4439..4458)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5252..5653,1..187)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
       61 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      241 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
      301 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      361 agtggccaac tccatcacta ggggttcctg cggccgcacg cgtgagggcc tatttcccat
      421 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
      481 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      541 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
      601 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggaaccgg
      661 agagtaggtc gggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
      721 acttgaaaaa gtggcaccga gtcggtgctt ttttgctagc gagggcctat ttcccatgat
      781 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
      841 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
      901 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
      961 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg cctcttttca
     1021 atctgactgg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1081 tgaaaaagtg gcaccgagtc ggtgcttttt taccggttct agactgcaga gggccctgcg
     1141 tatgagtgca agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga
     1201 ccgaccccga cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat
     1261 cagagagggg gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac
     1321 cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag
     1381 gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg
     1441 tccgcgccgc cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg
     1501 ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca
     1561 gcggaggagt cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc
     1621 gacatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     1681 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     1741 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     1801 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     1861 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     1921 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     1981 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     2041 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     2101 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     2161 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     2221 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     2281 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa ggaattctcc
     2341 ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca cctcgacagc
     2401 cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc agcgctaccg
     2461 ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc ctctgaagat
     2521 gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat gctgcggtac
     2581 accaacgggc cacctcccac ctaggatatc aagcttatcg ataccgagcg ctgctcgaga
     2641 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     2701 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     2761 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     2821 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     2881 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     2941 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     3001 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     3061 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     3121 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     3181 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     3241 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     3301 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3361 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3421 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3481 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3541 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3601 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3661 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3721 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3781 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3841 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3901 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3961 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4021 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4081 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4141 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4201 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4261 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4321 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4381 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4441 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4501 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4561 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4621 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4681 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4741 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4801 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4861 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4921 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4981 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5041 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5101 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5161 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5221 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5281 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5341 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     5401 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5461 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5521 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5581 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5641 agctatgaga aag
//
