LOCUS       sequence-459838-        6290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ple155-driven mRuby2, also encoding U6-driven sgRNA cassette, with 
            SapI sites to clone crRNA sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6290)
  AUTHORS   Coughlin GM, Borsos M, Barcelona BH, Appling N, Mayfield AMH, Mackey
            ED, Eser RA, Jackson CR, Chen X, Kumar SR, Gradinaru V
  TITLE     Spatial genomics of AAV vectors reveals mechanism of transcriptional
            crosstalk that enables targeted delivery of large genetic cargo.
  JOURNAL   Nat Biotechnol. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4.
  PUBMED    40113953
REFERENCE   2  (bases 1 to 6290)
  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: "Nat 
            Biotechnol. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4."
COMMENT     Sequence Label: 
            pAAV.U6-sasgRNA(SapI)_Ple155-CI-mRuby2-W3SL_BbsI(GGA)
FEATURES             Location/Qualifiers
     source          1..6290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     promoter        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     intron          2890..3022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      3029..3038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3035..3745
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(3810..3830)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4245..4385
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4245..4374
                     /label=AAV2 ITR
     rep_origin      complement(4447..5035)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4527..4546)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5206..5997,5998..6066))
                     /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     5829..5848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6067..6171)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6239..6257
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc ggaagagcga gctcttctgt tagagtactc tggaaacaga atctactcta
     1141 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt tggccgcctc
     1201 gagtcagcag attgaagaag ccctcctggt ctggggagcc cgcctgggga cagactcgct
     1261 cagtctggtt ggcccttcag cttgggggcc cctccccaaa cttcctagcc agcttgctca
     1321 caccctgacc ccggggcctg ccgtccccac ttcctcagct ctcaccatgg tccccgccga
     1381 tcctctctgc agagctgttc tgaatgagac atgagtctcc ttcccaatcc cggcccccgc
     1441 cccaggggcc ctggccagca gtgccacttc acgtggtacc gcttcaaggg acaggctccg
     1501 atgcgtgtcc cgccgtccta gattggggcc tgatgagtgt ggcctgggag ctgggacacg
     1561 aatcagggaa acatggccca ggagctaccc ccaggtccca gcatctccca tcaatagggg
     1621 tccacacgga gagccctgcc ctctgccctg gggcctggca ctcagacccc caagcccacc
     1681 agcccctttc tacagccaca actgggtcag ggggtcctag gagactcact cgttaattag
     1741 gtgccctaca aactaattag tcttgtcaat catgggctct gagaccttga gctgggggtg
     1801 gggtgggggc agggccctct cacctcggca caggggcctg agccttcctc cgtcttctcc
     1861 tcctgatccg gacacttcat tggcatagag ggagagagtg tgaacttggc cctttgtgga
     1921 acagaggagg ctcgggcaga ggtggtgata gtgcagccca ttcattctga gatgaaactt
     1981 ccactggttt ccgtaaagac gtcttgggga gggaagggaa ggggatgggg acctcccagt
     2041 ggtatcccct gcttgggcac tgagggaaag ccacagtggc tcggggtaaa aggcagggac
     2101 atcctctccc cgcctgcctc tgtccccagg gagtctcgcc tcctgttccc acctggggct
     2161 agggtgatag aggagaggag atagctcaac ctggcattta ggtggtgtgg gaacaggaga
     2221 ccccagactt tcttgttttg gggtctgggg caggcaacca ggctccaggg acagtgagtt
     2281 gaaggaaggg tggctgggag accccttgac ttgctgccaa ggagacagag ctggagctag
     2341 ggtggcgggt ggtgtctgag gcaggtgcag agagggaggg agggaagggg cctttgactc
     2401 caacctcctt tttctttacc gactgcaggt ggcagctgcc cttccaggag ccagtggggg
     2461 aacctgggtg gctgggtggg gacacctgca agtcctccct aagccagcta ccaccctaca
     2521 ctgttggcct cccttctcca actgtgggga cgctgctcag gccttttgtg acatcacacc
     2581 tgagagtccc tggggtccag tcattgctgc tgggcacagc gaggtccaag ctcaggtcgc
     2641 cctgccccct acccaccatg ccagatccag catcgttgtg ggcaaacaat tatctggatg
     2701 atctttatgg ggcttaagct tgggtgggag cagatggggc atgagctggg gatttgggga
     2761 tggggggaat ccacaccccc acgtcctgga cgtttaaaag gccctctctg gcactgggcc
     2821 ggggcagagg ccagcagaaa agtgactgga gtccaggcgc gccgaagttg gtcgtgaggc
     2881 actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc
     2941 ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc
     3001 cactttgcct ttctctccac agaccggtgc caccatggtg agcaagggag aggaactcat
     3061 caaggagaac atgcgcatga aggtagtcat ggaagggtct gtgaatgggc atcagttcaa
     3121 gtgcacaggg gaaggtgaag ggaatcccta catgggtact cagaccatga ggatcaaggt
     3181 tattgaaggt ggccctctcc cgtttgcctt cgacattctt gccactagct tcatgtatgg
     3241 cagtcggacc ttcatcaagt accccaaagg cattccagac ttcttcaaac agtcctttcc
     3301 agagggcttc acatgggaaa gggtgacccg ttacgaggat ggtggtgtgg tcacagtcat
     3361 gcaggacact agtctggagg acggatgtct ggtctatcac gtgcaagtga gaggggtgaa
     3421 ctttccctcc aatgggcctg tgatgcagaa gaaaaccaaa gggtgggagc caaacacaga
     3481 gatgatgtat cctgcagatg gaggactgag aggctatacg cacatggctc tgaaggttga
     3541 cggaggaggc cacttgtcat gctcatttgt gaccacgtac aggagcaaga aaactgtcgg
     3601 caacatcaaa atgcctggca tacatgcggt tgatcaccgc ttggaacgac tggaggagtc
     3661 tgacaacgag atgtttgtgg tacaacggga acatgcagtt gccaagtttg ctggccttgg
     3721 cggaggaatg gatgagctgt acaaatgaga attcataatc aacctctgga ttacaaaatt
     3781 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3841 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3901 tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3961 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgtttatttg tgaaatttgt
     4021 gatgctattg ctttatttgt aaccatctag ctttatttgt gaaatttgtg atgctattgc
     4081 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     4141 tatgtttcag gttcaggggg agatgtggga ggttttttaa acaagtgtct ggtgagggtc
     4201 ttcggtacca agaagaccta gtcacgtcag gtaccttggg ccgcaggaac ccctagtgat
     4261 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4321 cgcccgacgc ccgggctttg cccgagcggc ctcagtgagc gagcgagcgc gcagctgcct
     4381 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4441 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4501 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4561 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4621 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4681 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4741 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4801 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4861 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4921 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4981 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5041 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5101 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5161 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5221 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5281 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5341 accgcggctt ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5401 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5461 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5521 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5581 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5641 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5701 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5761 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5821 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5881 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5941 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6001 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6061 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6121 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6181 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6241 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
//
