LOCUS       sequence-394123-        6166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NLS-mRuby3 in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6166)
  AUTHORS   Moffa JC, Bland IN, Tooley JR, Kalyanaraman V, Heitmeier M, Creed 
            MC, Copits BA
  TITLE     Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and 
            Genetically Encoded Tool Delivery in the Central and Peripheral 
            Nervous Systems.
  JOURNAL   eNeuro. 2024 Jul 5;11(7):ENEURO.0438-23.2024. doi: 
            10.1523/ENEURO.0438-23.2024. Print 2024 Jul.
  PUBMED    38871457
REFERENCE   2  (bases 1 to 6166)
  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"; doi: 
            "10.1523/ENEURO.0438-23.2024"; journalName: "eNeuro"; date: 
            "2024-07-5- 5"; volume: "11"; issue: "7"; pages: 
            "ENEURO.0438-23.2024"
COMMENT     Sequence Label: pX552-DIO-NLS-mRuby3(with gRNA scaffold)
FEATURES             Location/Qualifiers
     source          1..6166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        459..1637
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          690..1628
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1585..1605
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1668..1701
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1764..1797
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1811..2521)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             complement(2531..2551)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2554..2563
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2574..2607)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2678..2711)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2769..3245
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2968..2987)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3285..3425
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3285..3414
                     /label=AAV2 ITR
     rep_origin      3500..3955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3587..3606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3797..3818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3972..3991)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4091..4113
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4151..4169)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4237..4341
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4342..4410,4411..5202)
                     /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(4560..4579)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5373..5961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5862..5881
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6023..6152
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gaagagcgag ctcttctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagactgca
      361 gagggccatc gatagctttg caaagatgga taaagtttta aacagagagg aatctttgca
      421 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
      481 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
      541 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      601 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
      661 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
      721 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
      781 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
      841 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
      901 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
      961 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     1021 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     1081 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     1141 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     1201 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     1261 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     1321 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     1381 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1441 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1501 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1561 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1621 catttcaggt gtcgtgagga attggatcct ctagagtcga aattcccata acttcgtata
     1681 aagtatccta tacgaagtta tatcaaaata ggaagaccaa tgcttcacca tcgacccgaa
     1741 ttgccaagca tcaccatcga cccataactt cgtataatgt atgctatacg aagttatact
     1801 agctagccta cttgtacagc tcgtccatgc caccaccaag gttgctgtat ttggcaactg
     1861 ccacttctct ttgcactacg taggtttcat tgtcactctc ctcgatcctt tccaggcggt
     1921 gatcaacggc atggacaccg ggcatcttga tgttcccgac ggtctttttt gacctgtaag
     1981 ttgtcacgaa gttgcagtgc agatggccac caccatcaac tttcagtgcg atgtcagtgt
     2041 atcctctcag accaccatct gctggataca tcatctctgt attaggctcc caacccttgg
     2101 tcttcttctg catcacggga ccattggagg gaaagtttac ccctctgacc ttgacgttgt
     2161 agacgagctc gccatcctca aggctggtgt cctgcgtgac ggtgacgact ccaccatctt
     2221 cgtatctcgt aactctttcc caagtaaaac cctcaggaaa ggactgttta aagaaatcag
     2281 ggatgtcggc cgggtacttg ataaaggtac ggctgccata catgaacgac gtggcaagaa
     2341 tgtcaaaggc aaatggcagg ggtcctccct cgatgacttt gatcctcatg gtttgcactc
     2401 cctcgtacgg tctgccttct ccttcacctg tgcatttgaa ttggtggccg ttgaccgaac
     2461 cttccatgac caccttcata cgcatatttt ccttgatcag ttcttcgccc ttagacacca
     2521 ttgggtcttc cactttcctc ttctttttgg gggccatggt ggcggtacct agtataactt
     2581 cgtataggat actttatacg aagttatcat tgggattctt cctattttga tccaagcatc
     2641 accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata gcatacatta
     2701 tacgaagtta tgtccctcga agaggttcgc gaagcttatc gataccgagc gctgctcgag
     2761 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     2821 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     2881 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     2941 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     3001 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     3061 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     3121 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     3181 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     3241 tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     3301 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3361 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3421 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3481 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3541 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     3601 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3661 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     3721 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3781 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     3841 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     3901 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     3961 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4021 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4081 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4141 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4201 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4261 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4321 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4381 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4441 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4501 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4561 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4621 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4681 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4741 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4801 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4861 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4921 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4981 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5041 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5101 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5161 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5221 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5281 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5341 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5401 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5461 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5521 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5581 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5641 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5701 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5761 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5821 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5881 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5941 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6001 tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc
     6061 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
     6121 gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgt
//
