LOCUS       sequence-455278-        6363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus sgRNAs backbone along with mCherry expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6363)
  AUTHORS   Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A,
            Hardin A, Eckalbar WL, Vaisse C, Ahituv N
  TITLE     CRISPR-mediated activation of a promoter or enhancer rescues obesity
            caused by haploinsufficiency.
  JOURNAL   Science. 2018 Dec 13. pii: science.aau0629. doi: 
            10.1126/science.aau0629.
  PUBMED    30545847
REFERENCE   2  (bases 1 to 6363)
  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: "Science. 
            2018 Dec 13. pii: science.aau0629. doi: 10.1126/science.aau0629."
COMMENT     Sequence Label: pAAV-U6-sasgRNACMV- mCherry-WPREpA
FEATURES             Location/Qualifiers
     source          1..6363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(211..222)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(331..347)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(332..348)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(393..410)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    399..606
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   619..748
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(810..1398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(890..909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1569..2360,2361..2429))
                     /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     2192..2211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2430..2534)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2602..2620
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2658..2680)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2780..2799
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2816..3271)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2953..2974)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3165..3184
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3346..3486
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3357..3486
                     /label=AAV2 ITR
     protein_bind    complement(3729..3762)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     3766..3786
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        3871..3946
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        4071..4374
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4375..4578
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4529..4549
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4612..5208
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4612..4631)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5108..5128
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5218..5271
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      5278..5287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5284..5994
                     /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(5429..5447)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5697..5716
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(6014..6047)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(6103..6363,1..328)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6156..6176)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
       61 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
      121 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
      181 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
      241 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
      301 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga
      361 aaaacatgga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
      421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
      481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
      541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
      601 tggggagcta gaggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
      661 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc ggcctcagtg
      721 agcgagcgag cgcgcagctg cctgcaggac atgtgagcaa aaggccagca aaaggccagg
      781 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
      841 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
      901 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
      961 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1021 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1081 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1141 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1201 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1261 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1321 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1381 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1441 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1501 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     1561 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     1621 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     1681 tctggcccca gtgctgcaat gataccgcgg cttccacgct caccggctcc agatttatca
     1741 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     1801 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     1861 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     1921 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     1981 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2041 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2101 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2161 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2221 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2281 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2341 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2401 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2461 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2521 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     2581 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     2641 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2701 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     2761 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataaaatt
     2821 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2881 aaccaataga ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagatagag
     2941 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3001 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     3061 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3121 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3181 ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3241 gccgcgctta atgcgccgct acagggcgcg tactatggtt gctttgacgt atgcggtgtg
     3301 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgcccctgc aggcagctgc
     3361 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc
     3421 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     3481 gttcctgcgg ccgcagcaga gatccagttt ggttagtacc gggcccgctc tagagatccg
     3541 acgcgccatc tctaggcccg cgccggcccc ctcgcacgga cttgtgggag aagctcggct
     3601 actcccctgc cccggttaat ttgcatataa tatttcctag taactataga ggcttaatgt
     3661 gcgataaaag acagataatc tgttcttttt aatactagct acattttaca tgataggctt
     3721 ggatttctat aacttcgtat agcatacatt atacgaagtt ataaacagca caaaaggaaa
     3781 ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aagtatccct tggagaacca
     3841 ccttgttggt gctgactgct acatagccaa gttatagtac tctggaaaca gaatctacta
     3901 taacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt tttctcgagt
     3961 actaggatcc attaggcggc cgcgtggata accgtattac cgccatgcat tagttattaa
     4021 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     4081 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     4141 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     4201 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     4261 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     4321 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     4381 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     4441 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     4501 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     4561 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcccac catgaccgag
     4621 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc
     4681 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc
     4741 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4801 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4861 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4921 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4981 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     5041 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     5101 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     5161 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atcgggagag
     5221 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc accggtcgcc
     5281 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     5341 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     5401 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     5461 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     5521 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     5581 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     5641 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     5701 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     5761 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     5821 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     5881 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     5941 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaagaattc
     6001 gtcgagggac ctaataactt cgtatagcat acattatacg aagttataca tgtttaaggg
     6061 ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac
     6121 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     6181 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     6241 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     6301 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     6361 acc
//
