LOCUS       sequence-296062-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of GluA1: C-terminal (amino acid position: STOP 
            codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7232)
  AUTHORS   Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, 
            Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD
  TITLE     ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope 
            tagging of endogenous proteins in neurons.
  JOURNAL   PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 
            10.1371/journal.pbio.3000665.
  PUBMED    32275651
REFERENCE   2  (bases 1 to 7232)
  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: "PLoS 
            Biol."; date: "2020-04-10"; volume: "18(4):e3000665. doi"; pages: " 
            10.1371/journal.pbio.3000665"
COMMENT     Sequence Label: pAAV ORANGE Gria1-HaloTag KI
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        193..441
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     193..213
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     364..383
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        462..537
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             690..1580
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     promoter        1655..2833
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1886..2824
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2781..2801
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2840..3550
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MQVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN
                     FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGAL
                     KGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER
                     AEGRHSTGGMDELYK"
     primer_bind     complement(2988..3006)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3256..3275
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3867..4343
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4066..4085)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(4665..5253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4745..4764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5424..6215,6216..6284))
                     /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     6047..6066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6285..6389)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6457..6475
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6513..6535)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6635..6654
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6721..7176)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6858..6879)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7070..7089
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgctattacg ccaggctgca gggggggggg gnnngnnggc cactccctct ctgcgcgctc
       61 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      181 cagatcccat gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag
      241 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
      301 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      361 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      421 ttgtggaaag gacgaaacac cgggagccac aggattgtaa cgttttagag ctagaaatag
      481 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      541 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg
      601 ccaattctgc agacaaatgg ctctagaagc ttacccgtta caatcctgtg gctcccggcg
      661 ctagcggtag cggctccagt agatctggga tggcagaaat cggtactggc tttccattcg
      721 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
      781 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
      841 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
      901 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
      961 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     1021 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1081 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1141 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1201 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1261 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1321 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1381 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1441 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1501 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     1561 ccacgctcga gatttccggc taagcgctcc cgttacaatc ctgtggctcc cacgcgccgc
     1621 tagcggtacc ggatcctacg tatctagagt cgacggctcc ggtgcccgtc agtgggcaga
     1681 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     1741 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1801 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1861 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
     1921 ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat
     1981 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg
     2041 agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg
     2101 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
     2161 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca
     2221 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc
     2281 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg
     2341 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc
     2401 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
     2461 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg
     2521 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc
     2581 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc
     2641 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga
     2701 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
     2761 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt
     2821 tcaggtgtcg tgagccacca tgcaagtgag caagggcgag gaggataaca tggccatcat
     2881 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2941 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     3001 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     3061 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     3121 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     3181 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     3241 cttcccctcc gacggccccg taatgcagaa gaagactatg ggctgggagg cctcctccga
     3301 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     3361 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     3421 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     3481 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     3541 gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca gcaggatgcc
     3601 ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa gggtgatcag
     3661 ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct gcctgctacc
     3721 tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat ccttctaccc
     3781 catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcgatatca agcttatcga
     3841 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     3901 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     3961 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4021 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     4081 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     4141 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     4201 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     4261 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     4321 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     4381 gatctgagga acccctagtg atggagttgg ccactccctc tctgcagcct gcattaatga
     4441 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4501 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4561 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4621 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4681 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4741 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4801 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4861 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4921 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4981 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5041 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5101 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5161 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5221 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5281 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5341 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5401 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5461 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5521 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5581 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5641 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5701 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5761 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5821 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5881 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5941 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6001 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6061 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6121 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6181 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6241 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6301 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6361 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6421 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6481 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     6541 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     6601 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     6661 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     6721 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6781 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     6841 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6901 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     6961 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     7021 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7081 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7141 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     7201 ctgttgggaa gggcgatcgg tgcgggcctc tt
//
