LOCUS       sequence-452436-        5541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pU6 (human U6) expression of SpCas9 sgRNA targeting RHO P23H 
            mutation and pCMV expression of mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5541)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 5541)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pAAV-U6-SpCas9_gRNA(RHO-P23H)-CMV-mTagBFP2_(RA...
FEATURES             Location/Qualifiers
     source          1..5541
                     /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        852..1100
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     852..872
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1023..1042
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1121..1196
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1209..1588
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1589..1792
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1743..1763
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1789..1813
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1928..2060
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2043..2062
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      2117..2126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2123..2833
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    2883..3471
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2936..2956)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3354..3365)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3512..3633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3549..3568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3603..3622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3704..3844
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3704..3833
                     /label=AAV2 ITR
     rep_origin      3919..4374
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4006..4025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4216..4237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4391..4410)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4510..4532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4570..4588)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4643..4662)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4654..5463
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4721..4740)
                     /label=Kan-R
                     /note="Kanamycin 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 gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg
      841 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      901 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      961 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1021 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     1081 tgtggaaagg acgaaacacc gtagtactgt gggtactcga gttttagagc tagaaatagc
     1141 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1201 tttctagcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     1261 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1321 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1381 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1441 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1501 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1561 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     1621 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     1681 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     1741 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
     1801 gaaccgtcag atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc
     1861 agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agaagttggt cgtgaggcac
     1921 tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt
     1981 gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca
     2041 ctttgccttt ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag
     2101 tacttaatta accggtgcca ccatggtgtc taagggcgaa gagctgatta aggagaacat
     2161 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     2221 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     2281 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     2341 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     2401 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     2461 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     2521 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     2581 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     2641 tctgatcgca aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     2701 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     2761 ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg
     2821 gcacaaactt aattactcag atctcgagct caagtagctt aagaattcat cagcttatcg
     2881 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2941 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3001 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3061 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3121 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3181 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3241 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3301 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3361 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3421 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3481 gagcgctcta gagctcgctg atcagcctcg aaacttgttt attgcagctt ataatggtta
     3541 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3601 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccag caggctttcc
     3661 taggttctca cgtgcggacc gaggctgcag cgtcgtcctc cctaggaacc cctagtgatg
     3721 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     3781 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     3841 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3901 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3961 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4021 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4081 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4141 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4201 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     4261 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     4321 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     4381 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4441 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4501 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4561 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgaac aataaaactg
     4621 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     4681 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     4741 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     4801 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     4861 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     4921 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     4981 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     5041 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     5101 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     5161 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     5221 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     5281 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     5341 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     5401 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     5461 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5521 agaaaagatc aaaggatctt c
//
