LOCUS       sequence-369120-        6265 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Contains HaloTag to be inserted into the NTD of Gria1 (via HITI), 
            single guide RNA to target Cas9 to Gria1 under control of the U6 
            promoter, and miRFP670 under control of human synapsin promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6265)
  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 6265)
  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: "Elife."; 
            date: "2024-08-15"; volume: "13:e80622. doi"; pages: " 
            10.7554/eLife.80622"
COMMENT     Sequence Label: px552-sg-gria1-HT
FEATURES             Location/Qualifiers
     source          1..6265
                     /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"
     promoter        1548..1995
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      2009..2018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2015..2962
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    3012..3488
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3211..3230)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3528..3668
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3528..3657
                     /label=AAV2 ITR
     rep_origin      3743..4198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3830..3849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4040..4061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4215..4234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4334..4356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4394..4412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4480..4584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4585..4653,4654..5445)
                     /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(4803..4822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5616..6204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6105..6124
                     /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 ccggttctgg tagcggctcc tccaccctgg
     1501 tatggtctcg ggaggctagc tctagactgc agagggccct gcgtatgagt gcaagtgggt
     1561 tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg
     1621 gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga
     1681 aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg
     1741 cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc
     1801 gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc
     1861 ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc
     1921 tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg
     1981 tgcctgagag cgcagtcgag aaggtaccgc caccatggta gcaggtcatg cctctggcag
     2041 ccccgcattc gggaccgcct ctcattcgaa ttgcgaacat gaagagatcc acctcgccgg
     2101 ctcgatccag ccgcatggcg cgcttctggt cgtcagcgaa catgatcatc gcgtcatcca
     2161 ggccagcgcc aacgccgcgg aatttctgaa tctcggaagc gtactcggcg ttccgctcgc
     2221 cgagatcgac ggcgatctgt tgatcaagat cctgccgcat ctcgatccca ccgccgaagg
     2281 catgccggtc gcggtgcgct gccggatcgg caatccctct acggagtact gcggtctgat
     2341 gcatcggcct ccggaaggcg ggctgatcat cgaactcgaa cgtgccggcc cgtcgatcga
     2401 tctgtcaggc acgctggcgc cggcgctgga gcggatccgc acggcgggtt cactgcgcgc
     2461 gctgtgcgat gacaccgtgc tgctgtttca gcagtgcacc ggctacgacc gggtgatggt
     2521 gtatcgtttc gatgagcaag gccacggcct ggtattctcc gagtgccatg tgcctgggct
     2581 cgaatcctat ttcggcaacc gctatccgtc gtcgactgtc ccgcagatgg cgcggcagct
     2641 gtacgtgcgg cagcgcgtcc gcgtgctggt cgacgtcacc tatcagccgg tgccgctgga
     2701 gccgcggctg tcgccgctga ccgggcgcga tctcgacatg tcgggctgct tcctgcgctc
     2761 gatgtcgccg tgccatctgc agttcctgaa ggacatgggc gtgcgcgcca ccctggcggt
     2821 gtcgctggtg gtcggcggca agctgtgggg cctggttgtc tgtcaccatt atctgccgcg
     2881 cttcatccgt ttcgagctgc gggcgatctg caaacggctc gccgaaagga tcgcgacgcg
     2941 gatcaccgcg cttgagagct aagaattcga tatcaagctt atcgataccg agcgctgctc
     3001 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     3061 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     3121 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     3181 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     3241 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     3301 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     3361 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     3421 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     3481 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     3541 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3601 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3661 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3721 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3781 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     3841 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3901 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     3961 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4021 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     4081 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     4141 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     4201 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4261 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4321 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4381 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4441 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6241 ttttgctggc cttttgctca catgt
//
