LOCUS       sequence-425080-        5971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Grin1 Control gRNA in a Cre dependent GCaMP8f vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5971)
  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 5971)
  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-hSyn-DIO-GCaMP8f_Grin1TTT gRNA
FEATURES             Location/Qualifiers
     source          1..5971
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        379..826
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     primer_bind     898..917
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    952..985
                     /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    1048..1081
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1666..1685
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1925..1946
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2052..2073)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2355..2372)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2382..2415)
                     /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(2486..2519)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2574..3050
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2773..2792)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3090..3230
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3090..3219
                     /label=AAV2 ITR
     rep_origin      3305..3760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3392..3411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3602..3623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3777..3796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3896..3918
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3956..3974)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4042..4146
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4147..4215,4216..5007)
                     /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(4365..4384)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5178..5766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5667..5686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5828..5957
                     /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 cacgagcaga tgttcctttg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagactg
      361 cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg
      421 tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat
      481 tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact
      541 gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg
      601 cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg
      661 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga
      721 gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca
      781 gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga gaaggtaccg
      841 gatctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
      901 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attggatccc cataacttcg
      961 tataaagtat cctatacgaa gttatatcaa aataggaaga ccaatgcttc accatcgacc
     1021 cgaattgcca agcatcacca tcgacccata acttcgtata atgtatgcta tacgaagtta
     1081 tgaattcctg caggagctca ctagtttcac ttcgctgtca tcatttgtac aaactcttcg
     1141 tagtttacct gaccatcccc atcgatgtct gcttccctga tcatttcatc aacctcttca
     1201 tctgttaact tctctccaag gtttgtcatc acgtggcgaa gctctgctgc actgatgtag
     1261 ccattgccat ccttatcaaa cacaccgaac gcttctctaa tttcttcttc cgtgtccctg
     1321 tatttcattt ttcttgccat cattgtcagg aactcaggga agtcgattgt gccgtcaccg
     1381 tcggcatcta cttcattgat catgtcctgc agctctgctt ctgtggggtt cagtccgaga
     1441 gaccgcatca ccgtccccag ctccttggtt gttattgtcc catccccgtc cttgtcaaat
     1501 agggagaaag cctctttaaa ttctgcgatc tgctctccag tcagttggtc cggcaggttg
     1561 tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg
     1621 atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg
     1681 tcgtccttga agaagatggt gcgctcctgg atgtagcctt cgggcatggc ggacttgaag
     1741 aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg
     1801 gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg
     1861 gtcagcttgc cgtaggtggc atcgccctca ccctcgccgg acacgctgaa cttgtggccg
     1921 tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc
     1981 ttgctcacca tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc gagagtgatc
     2041 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttc
     2101 gaaagtttgg actccacgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     2161 atgggggtgt tctgctggta gtggtaggcg agctgcacgc cgccgtcctc gatgttgtgg
     2221 cggatgtgga agttcgcctt gatgccgttc ttctgcttgt cggccttgat atagacgttg
     2281 atcttcaggc ccatcaggcg ggcgatgatc ttcacggcgg tggccacctc cttgaaggtc
     2341 ttcttgcgac gcgtgtgatg atggtgatga tgcatgatat cataacttcg tataggatac
     2401 tttatacgaa gttatcattg ggattcttcc tattttgatc caagcatcac catcgaccct
     2461 ctagtccaga tctcaccatc gacccataac ttcgtatagc atacattata cgaagttatg
     2521 tccctcgaag aggttcaagc ttatcgatac cgagcgctgc tcgagagatc tacgggtggc
     2581 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     2641 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     2701 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     2761 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     2821 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     2881 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     2941 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     3001 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt
     3061 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     3121 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     3181 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     3241 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     3301 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3361 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     3421 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3481 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     3541 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3601 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     3661 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     3721 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     3781 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     3841 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3901 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     3961 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     4021 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4081 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4141 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4201 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4261 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4321 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4381 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4441 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4501 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4561 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4621 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4681 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4741 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4801 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4861 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4921 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4981 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5041 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5101 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5161 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5221 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5281 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5341 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5401 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5461 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5521 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5581 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5641 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5701 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5761 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5821 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
     5881 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
     5941 catcactagg ggttcctgcg gccgcacgcg t
//
