LOCUS       sequence-457988-        5283 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CTRL knockdown gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5283)
  TITLE     AAV-CRISPR/Cas9 vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5283)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-u6-gRNA(CTRL)-hSyn-mCherry
FEATURES             Location/Qualifiers
     source          1..5283
                     /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        380..827
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             859..1569
                     /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(1004..1022)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1272..1291
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1886..2362
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2085..2104)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2402..2542
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2402..2531
                     /label=AAV2 ITR
     rep_origin      2617..3072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2704..2723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2914..2935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3089..3108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3208..3230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3268..3286)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3354..3458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3459..3527,3528..4319)
                     /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(3677..3696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4490..5078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4979..4998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5140..5269
                     /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 gtgagcgagt aacaacccgt gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagact
      361 gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg
      421 gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa
      481 ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac
      541 tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc
      601 gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg
      661 gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg
      721 agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc
      781 agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg agaaggtacc
      841 ggatcctcta gagtcgacat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
      901 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
      961 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     1021 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     1081 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     1141 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     1201 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     1261 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     1321 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     1381 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     1441 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     1501 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     1561 tacaagtagt ccggactcag atctcgagag gaggaggagg agacagacag caggatgccc
     1621 cacctcgaca gccccggcag ctcccagccg agacgctcct tcctctcaag ggtgatcagg
     1681 gcagcgctac cgttgcagct gcttctgctg ctgctgctgc tcctggcctg cctgctacct
     1741 gcctctgaag atgactacag ctgcacccag gccaacaact ttgcccgatc cttctacccc
     1801 atgctgcggt acaccaacgg gccacctccc acctaggaat tcgatatcaa gcttatcgat
     1861 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     1921 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     1981 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     2041 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     2101 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     2161 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     2221 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     2281 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     2341 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     2401 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     2461 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     2521 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     2581 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     2641 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     2701 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2761 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2821 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2881 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2941 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     3001 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3061 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3121 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3241 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3301 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3361 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3421 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3481 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3541 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3601 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3661 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3721 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3781 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3841 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3901 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3961 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4021 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4081 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4141 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4201 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4261 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4321 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4381 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4501 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4561 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4621 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4681 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4741 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4801 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4861 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4921 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4981 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5041 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5101 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     5161 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     5221 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg
     5281 cgt
//
