LOCUS       sequence-422890-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Dicer gRNA in a Cre dependent mcherry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  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 6096)
  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-EF1a-DIO-mCherry_Dicer gRNA
FEATURES             Location/Qualifiers
     source          1..6096
                     /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        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        462..1640
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          693..1631
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1588..1608
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1671..1704)
                     /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(1755..1788)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1800..2510)
                     /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(2078..2097)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2347..2365
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    2532..2565
                     /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    2616..2649
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2699..3175
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2898..2917)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3215..3355
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3215..3344
                     /label=AAV2 ITR
     rep_origin      3430..3885
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3517..3536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3727..3748
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3902..3921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4021..4043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4081..4099)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4167..4271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4272..4340,4341..5132)
                     /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(4490..4509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5953..6082
                     /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 gacccattgg tgaggaagca gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagact
      361 gcagagggcc atcgatagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt
      421 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
      481 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
      541 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      601 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      661 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
      721 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
      781 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
      841 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
      901 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
      961 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     1021 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     1081 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     1141 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     1201 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     1261 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     1321 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     1381 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     1441 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     1501 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     1561 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     1621 ttccatttca ggtgtcgtga ggaattggat cctctagagt cgactccgga ataacttcgt
     1681 ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg ctattagcaa
     1741 tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg cgcgccttat
     1801 tacttgtaca gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt
     1861 tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag
     1921 gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag
     1981 tggccgccgt ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg
     2041 gggtacatcc gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg
     2101 tcggagggga agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg
     2161 gaggagtcct gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac
     2221 ttgaagccct cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg
     2281 taggccttgg agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg
     2341 ccacccttgg tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc
     2401 tcgccctcga tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc
     2461 atgaactcct tgatgatggc catgttatcc tcctcgccct tgctcaccat tggcccgggg
     2521 ctagcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga
     2581 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata
     2641 cgaagttatg aattcgatat caagcttatc gataccgagc gctgctcgag agatctacgg
     2701 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     2761 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     2821 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     2881 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     2941 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     3001 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     3061 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     3121 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     3181 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     3241 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     3301 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     3361 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     3421 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3481 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     3541 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3601 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     3661 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3721 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     3781 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     3841 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     3901 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     3961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4081 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4141 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4201 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4261 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4321 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4381 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4441 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4501 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4561 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4621 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4681 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4741 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4801 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4861 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4921 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4981 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5041 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5101 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5161 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
     6001 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     6061 aactccatca ctaggggttc ctgcggccgc acgcgt
//
