LOCUS       sequence-369129-        5020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains HaloTag-SEP to be inserted into the NTD of Gria1 (via HITI)
            and single guide RNA to target Cas9 to Gria1 under control of the U6
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5020)
  AUTHORS   Wong VC, Houlihan PR, Liu H, Walpita D, DeSantis MC, Liu Z, O'Shea 
            EK
  TITLE     Plasticity-induced actin polymerization in the dendritic shaft 
            regulates intracellular AMPA receptor trafficking.
  JOURNAL   Elife. 2024 Aug 15;13:e80622. doi: 10.7554/eLife.80622.
  PUBMED    39146380
REFERENCE   2  (bases 1 to 5020)
  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"; journalName: "Elife."; 
            date: "2024-08-15"; volume: "13:e80622. doi"; pages: " 
            10.7554/eLife.80622"
COMMENT     Sequence Label: px552-sg-gria1-HT-SEP
FEATURES             Location/Qualifiers
     source          1..5020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             555..575
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             591..1475
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             1488..2198
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1515..1543)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2138..2158
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     repeat_region   2283..2423
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2283..2412
                     /label=AAV2 ITR
     rep_origin      2498..2953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2585..2604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2795..2816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2970..2989)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3089..3111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3149..3167)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3235..3339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3340..3408,3409..4200)
                     /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(3558..3577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4371..4959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4860..4879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accctcccga gaccatacca ggggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag taccggtcaa ttgccaccct ggtatggtct cgggagggtg
      541 gcggaggttc tgtggaggat ctgtactttc agagcgataa cgatggatcc gaaatcggta
      601 ctggctttcc attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg
      661 atgttggtcc gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct
      721 acgtgtggcg caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc
      781 tgatcggtat gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc
      841 gcttcatgga tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg
      901 actggggctc cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta
      961 ttgcatttat ggagttcatc cgccctatcc cgacctggga cgaatggcca gaatttgccc
     1021 gcgagacctt ccaggccttc cgcaccaccg acgtcggccg caagctgatc atcgatcaga
     1081 acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga
     1141 tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc
     1201 caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca
     1261 tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc
     1321 tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg
     1381 acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga
     1441 tcgcgcgctg gctgtctact ctggagattt ccggtggcgg aggcgggagc aaaggagaag
     1501 aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca
     1561 aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat
     1621 ttatttgcac tactggaaaa ctacctgttc cttggccaac acttgtcact actttaactt
     1681 atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac tttttcaaga
     1741 gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact
     1801 acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa
     1861 aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata
     1921 acgatcacca ggtgtacatc atggcagaca aacaaaagaa tggaatcaaa gctaacttca
     1981 aaattagaca caacattgaa gatggaggcg ttcaactagc agaccattat caacaaaata
     2041 ctccaattgg cgatgggccc gtccttttac cagacaacca ttacctgttt acaacttcta
     2101 ctctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag
     2161 ctgctgggat tacacatggc atggatgaac tatacaaatc tggtagcggc tcctccaccc
     2221 tggtatggtc tcgggaggct agctctagct ctagactgca gagggcccga ccgagcggcc
     2281 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     2341 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     2401 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     2461 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     2521 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     2581 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2641 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2701 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2761 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2821 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     2881 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2941 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3001 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3061 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3121 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3181 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3241 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3301 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3361 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3421 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3481 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3541 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3601 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3661 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3721 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3781 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3841 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3901 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3961 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4021 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4081 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4141 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4201 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4741 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4801 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4861 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4921 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4981 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
