LOCUS       sequence-339130-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  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 5646)
  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: sgLacZs
FEATURES             Location/Qualifiers
     source          1..5646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   249..378
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        393..641
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        664..739
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        752..1000
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1023..1098
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1138..1585
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1617..2324
                     /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(1762..1780)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2030..2049
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2641..3117
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2840..2859)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3157..3297
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3157..3286
                     /label=AAV2 ITR
     rep_origin      3372..3827
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3459..3478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3669..3690
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3844..3863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3963..3985
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4023..4041)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4109..4213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4214..4282,4283..5074)
                     /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(4432..4451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5245..5646,1..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 gggcgtcggg
      301 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
      361 ccatcactag gggttcctgc ggccgcacgc gtgagggcct atttcccatg attccttcat
      421 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
      481 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      541 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      601 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtgttcgca ttatccgaac
      661 catgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      721 agtggcaccg agtcggtgct tttttgctag cgagggccta tttcccatga ttccttcata
      781 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      841 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      901 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      961 tttcttggct ttatatatct tgtggaaagg acgaaacacc gcgcgatcgt aatcacccga
     1021 gtgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1081 gtggcaccga gtcggtgctt ttttaccggt tatagactgc agagggccct gcgtatgagt
     1141 gcaagtgggt tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc
     1201 cgacccactg gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag
     1261 ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac
     1321 agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct
     1381 gacgtcactc gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc
     1441 cgccgccggc ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga
     1501 ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg
     1561 agtcgtgtcg tgcctgagag cgcagtcgag aaggtaccgg atcctctaga gtcgacatgg
     1621 tgagcaaggg cgaggaggat aacatggcca tcatcgagga gttcatgcgc ttcaaggtgc
     1681 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1741 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     1801 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1861 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1921 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1981 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2041 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     2101 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     2161 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2221 tcaagttgga catcacctcc cacgacgagg actacaccat cgtggaacag tacgaacgcg
     2281 ccgagggccg ccactccacc ggcggcatgg acgagctgta caaggaattc tccggactca
     2341 gatctcgaga ggaggaggag gggacagaca gcaggatgcc ccacctcgac agccccggca
     2401 gctcccagcc gagacgctcc ttcctctcaa gggtgatcag ggcagcgcta ccgttgcagc
     2461 tgcttctgct gctgctgctg ctcctggcct gcctgctacc tgcctctgaa gatgactaca
     2521 gctgcaccca ggccaacaac tttgcccgat ccttctaccc catgctgcgg tacaccaacg
     2581 ggccacctcc cacctaggat atcaagctta tcgataccga gcgctgctcg agagatctac
     2641 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     2701 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     2761 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     2821 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     2881 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     2941 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     3001 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     3061 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     3121 attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg
     3181 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     3241 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     3301 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     3361 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3421 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     3481 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3541 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     3601 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3661 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     3721 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     3781 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     3841 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     3901 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3961 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4021 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4081 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4141 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4201 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4261 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4321 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4381 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4441 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     4501 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4561 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4621 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4681 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4741 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4801 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4861 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4921 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4981 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5041 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5101 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5161 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5221 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5281 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5341 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5401 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5461 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5521 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5581 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5641 agaaag
//
