LOCUS       sequence-459911-        5934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ple155-driven mRuby2, containing cassette for BbsI-based Golden Gate
            assembly (e.g. of sgRNA cassette(s)). Can be used as 'no guide' 
            control. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5934)
  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 5934)
  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.Ple155-CI-mRuby2-W3SL_BbsI(GGA)
FEATURES             Location/Qualifiers
     source          1..5934
                     /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
     intron          2534..2666
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      2673..2682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2679..3389
                     /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(3454..3474)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3889..4029
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3889..4018
                     /label=AAV2 ITR
     rep_origin      complement(4091..4679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4171..4190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4850..5641,5642..5710))
                     /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     5473..5492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5711..5815)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5883..5901
                     /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 cctcgagtca gcagattgaa gaagccctcc tggtctgggg agcccgcctg gggacagact
      901 cgctcagtct ggttggccct tcagcttggg ggcccctccc caaacttcct agccagcttg
      961 ctcacaccct gaccccgggg cctgccgtcc ccacttcctc agctctcacc atggtccccg
     1021 ccgatcctct ctgcagagct gttctgaatg agacatgagt ctccttccca atcccggccc
     1081 ccgccccagg ggccctggcc agcagtgcca cttcacgtgg taccgcttca agggacaggc
     1141 tccgatgcgt gtcccgccgt cctagattgg ggcctgatga gtgtggcctg ggagctggga
     1201 cacgaatcag ggaaacatgg cccaggagct acccccaggt cccagcatct cccatcaata
     1261 ggggtccaca cggagagccc tgccctctgc cctggggcct ggcactcaga cccccaagcc
     1321 caccagcccc tttctacagc cacaactggg tcagggggtc ctaggagact cactcgttaa
     1381 ttaggtgccc tacaaactaa ttagtcttgt caatcatggg ctctgagacc ttgagctggg
     1441 ggtggggtgg gggcagggcc ctctcacctc ggcacagggg cctgagcctt cctccgtctt
     1501 ctcctcctga tccggacact tcattggcat agagggagag agtgtgaact tggccctttg
     1561 tggaacagag gaggctcggg cagaggtggt gatagtgcag cccattcatt ctgagatgaa
     1621 acttccactg gtttccgtaa agacgtcttg gggagggaag ggaaggggat ggggacctcc
     1681 cagtggtatc ccctgcttgg gcactgaggg aaagccacag tggctcgggg taaaaggcag
     1741 ggacatcctc tccccgcctg cctctgtccc cagggagtct cgcctcctgt tcccacctgg
     1801 ggctagggtg atagaggaga ggagatagct caacctggca tttaggtggt gtgggaacag
     1861 gagaccccag actttcttgt tttggggtct ggggcaggca accaggctcc agggacagtg
     1921 agttgaagga agggtggctg ggagacccct tgacttgctg ccaaggagac agagctggag
     1981 ctagggtggc gggtggtgtc tgaggcaggt gcagagaggg agggagggaa ggggcctttg
     2041 actccaacct cctttttctt taccgactgc aggtggcagc tgcccttcca ggagccagtg
     2101 ggggaacctg ggtggctggg tggggacacc tgcaagtcct ccctaagcca gctaccaccc
     2161 tacactgttg gcctcccttc tccaactgtg gggacgctgc tcaggccttt tgtgacatca
     2221 cacctgagag tccctggggt ccagtcattg ctgctgggca cagcgaggtc caagctcagg
     2281 tcgccctgcc ccctacccac catgccagat ccagcatcgt tgtgggcaaa caattatctg
     2341 gatgatcttt atggggctta agcttgggtg ggagcagatg gggcatgagc tggggatttg
     2401 gggatggggg gaatccacac ccccacgtcc tggacgttta aaaggccctc tctggcactg
     2461 ggccggggca gaggccagca gaaaagtgac tggagtccag gcgcgccgaa gttggtcgtg
     2521 aggcactggg caggtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact
     2581 gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactga
     2641 catccacttt gcctttctct ccacagaccg gtgccaccat ggtgagcaag ggagaggaac
     2701 tcatcaagga gaacatgcgc atgaaggtag tcatggaagg gtctgtgaat gggcatcagt
     2761 tcaagtgcac aggggaaggt gaagggaatc cctacatggg tactcagacc atgaggatca
     2821 aggttattga aggtggccct ctcccgtttg ccttcgacat tcttgccact agcttcatgt
     2881 atggcagtcg gaccttcatc aagtacccca aaggcattcc agacttcttc aaacagtcct
     2941 ttccagaggg cttcacatgg gaaagggtga cccgttacga ggatggtggt gtggtcacag
     3001 tcatgcagga cactagtctg gaggacggat gtctggtcta tcacgtgcaa gtgagagggg
     3061 tgaactttcc ctccaatggg cctgtgatgc agaagaaaac caaagggtgg gagccaaaca
     3121 cagagatgat gtatcctgca gatggaggac tgagaggcta tacgcacatg gctctgaagg
     3181 ttgacggagg aggccacttg tcatgctcat ttgtgaccac gtacaggagc aagaaaactg
     3241 tcggcaacat caaaatgcct ggcatacatg cggttgatca ccgcttggaa cgactggagg
     3301 agtctgacaa cgagatgttt gtggtacaac gggaacatgc agttgccaag tttgctggcc
     3361 ttggcggagg aatggatgag ctgtacaaat gagaattcat aatcaacctc tggattacaa
     3421 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3481 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3541 cttgtataaa tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg
     3601 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat
     3661 ttgtgatgct attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta
     3721 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3781 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaacaagt gtctggtgag
     3841 ggtcttcggt accaagaaga cctagtcacg tcaggtacct tgggccgcag gaacccctag
     3901 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3961 aggtcgcccg acgcccgggc tttgcccgag cggcctcagt gagcgagcga gcgcgcagct
     4021 gcctgcagga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4081 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4141 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4201 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4261 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4321 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4381 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4441 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4501 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4561 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4621 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4681 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4741 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4801 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4861 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4921 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4981 tgataccgcg gcttccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5041 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5101 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5161 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5221 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5281 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5341 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5401 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5461 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5521 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5581 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5641 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5701 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5761 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5821 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5881 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtg
//
