LOCUS       sequence-425074-        5970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Grin 1 gRNA in a Cre dependent GCaMP8f vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5970)
  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 5970)
  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_Grin1 gRNA
FEATURES             Location/Qualifiers
     source          1..5970
                     /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        269..344
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        378..825
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     primer_bind     897..916
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    951..984
                     /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    1047..1080
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1665..1684
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1924..1945
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2051..2072)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2354..2371)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2381..2414)
                     /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(2485..2518)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2573..3049
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2772..2791)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3089..3229
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3089..3218
                     /label=AAV2 ITR
     rep_origin      3304..3759
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3391..3410)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3601..3622
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3776..3795)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3895..3917
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3955..3973)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4041..4145
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4146..4214,4215..5006)
                     /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(4364..4383)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5177..5765
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5666..5685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5827..5956
                     /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 cacagcagat gttccgcggt tttagagcta gaaatagcaa gttaaaataa
      301 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tctagactgc
      361 agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg gggtgggggt
      421 gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat tccccaaatt
      481 gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg
      541 ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc
      601 ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc ccttcccggc
      661 caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc gaggcgcgag
      721 ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag
      781 tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag cgcagtcgag aaggtaccgg
      841 atctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa
      901 cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttggatcccc ataacttcgt
      961 ataaagtatc ctatacgaag ttatatcaaa ataggaagac caatgcttca ccatcgaccc
     1021 gaattgccaa gcatcaccat cgacccataa cttcgtataa tgtatgctat acgaagttat
     1081 gaattcctgc aggagctcac tagtttcact tcgctgtcat catttgtaca aactcttcgt
     1141 agtttacctg accatcccca tcgatgtctg cttccctgat catttcatca acctcttcat
     1201 ctgttaactt ctctccaagg tttgtcatca cgtggcgaag ctctgctgca ctgatgtagc
     1261 cattgccatc cttatcaaac acaccgaacg cttctctaat ttcttcttcc gtgtccctgt
     1321 atttcatttt tcttgccatc attgtcagga actcagggaa gtcgattgtg ccgtcaccgt
     1381 cggcatctac ttcattgatc atgtcctgca gctctgcttc tgtggggttc agtccgagag
     1441 accgcatcac cgtccccagc tccttggttg ttattgtccc atccccgtcc ttgtcaaata
     1501 gggagaaagc ctctttaaat tctgcgatct gctctccagt cagttggtcc ggcaggttgt
     1561 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     1621 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     1681 cgtccttgaa gaagatggtg cgctcctgga tgtagccttc gggcatggcg gacttgaaga
     1741 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
     1801 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     1861 tcagcttgcc gtaggtggca tcgccctcac cctcgccgga cacgctgaac ttgtggccgt
     1921 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     1981 tgctcaccat gctccctccg gtaccgccct tgtacagctc gtccatgccg agagtgatcc
     2041 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttcg
     2101 aaagtttgga ctccacgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     2161 tgggggtgtt ctgctggtag tggtaggcga gctgcacgcc gccgtcctcg atgttgtggc
     2221 ggatgtggaa gttcgccttg atgccgttct tctgcttgtc ggccttgata tagacgttga
     2281 tcttcaggcc catcaggcgg gcgatgatct tcacggcggt ggccacctcc ttgaaggtct
     2341 tcttgcgacg cgtgtgatga tggtgatgat gcatgatatc ataacttcgt ataggatact
     2401 ttatacgaag ttatcattgg gattcttcct attttgatcc aagcatcacc atcgaccctc
     2461 tagtccagat ctcaccatcg acccataact tcgtatagca tacattatac gaagttatgt
     2521 ccctcgaaga ggttcaagct tatcgatacc gagcgctgct cgagagatct acgggtggca
     2581 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     2641 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     2701 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     2761 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     2821 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     2881 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     2941 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     3001 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     3061 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     3121 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     3181 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     3241 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3301 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3361 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3421 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3481 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3541 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3601 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     3661 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     3721 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3781 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3841 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3901 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3961 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4021 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4081 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4141 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4201 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4261 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4321 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4381 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4441 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4501 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4561 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4621 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4681 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4741 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4801 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4861 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4921 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4981 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5041 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5101 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5161 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5221 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5281 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5341 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5401 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5461 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5521 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5581 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5641 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5701 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5761 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5821 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg
     5881 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     5941 atcactaggg gttcctgcgg ccgcacgcgt
//
