LOCUS       sequence-296061-        7207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of PSD95: C-terminal (amino acid position: R721).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7207)
  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 7207)
  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 Dlg4-HaloTag KI
FEATURES             Location/Qualifiers
     source          1..7207
                     /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        463..538
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             666..1556
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIHPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     promoter        1630..2808
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1861..2799
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2756..2776
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2815..3525
                     /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(2963..2981)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3231..3250
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3842..4318
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4041..4060)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(4640..5228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4720..4739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5399..6190,6191..6259))
                     /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     6022..6041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6260..6364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6432..6450
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6488..6510)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6610..6629
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6696..7151)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6833..6854)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7045..7064
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgctattacg ccaggctgca gggggggggg gggggnnggc 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 cgaatcagag tctctctcgg gcgttttaga gctagaaata
      481 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      541 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      601 gccaattctg cagacaaatg gctctagaag cttaatcaga gtctctctcg ggcggggcta
      661 gcggaatggc agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg
      721 gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc
      781 acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc
      841 atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt
      901 atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag
      961 aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca
     1021 atccagagcg cgtcaaaggt attgcattta tggagttcat ccaccctatc ccgacctggg
     1081 acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc
     1141 gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc
     1201 gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc
     1261 gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg
     1321 tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt
     1381 tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc
     1441 tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc
     1501 cggacctgat cggcagcgag atcgcgcgct ggctgtccac gctcgagatt tccggcagcg
     1561 ctaaatcaga gtctctctcg ggcgggacgc gccgctagcg gtaccggatc ctacgtatct
     1621 agagtcgacg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     1681 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     1741 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     1801 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     1861 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     1921 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     1981 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2041 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2101 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2161 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2221 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2281 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2341 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2401 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2461 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     2521 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     2581 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     2641 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     2701 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     2761 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc caccatgcaa
     2821 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     2881 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2941 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3001 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     3061 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3121 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3181 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     3241 cagaagaaga ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     3301 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3361 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3421 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3481 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtccgg actcagatct
     3541 cgagaggagg aggaggagac agacagcagg atgccccacc tcgacagccc cggcagctcc
     3601 cagccgagac gctccttcct ctcaagggtg atcagggcag cgctaccgtt gcagctgctt
     3661 ctgctgctgc tgctgctcct ggcctgcctg ctacctgcct ctgaagatga ctacagctgc
     3721 acccaggcca acaactttgc ccgatccttc taccccatgc tgcggtacac caacgggcca
     3781 cctcccacct aggaattcga tatcaagctt atcgataccg agcgctgctc gagagatcta
     3841 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     3901 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     3961 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     4021 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     4081 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     4141 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     4201 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     4261 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4321 gattttgtag gtaaccacgt gcggaccgag cggccgatct gaggaacccc tagtgatgga
     4381 gttggccact ccctctctgc agcctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4441 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4501 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4561 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4801 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5041 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5281 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5341 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5401 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5461 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5521 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5581 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5641 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5701 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5761 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5821 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5881 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5941 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6001 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6061 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6361 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6421 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6481 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6541 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6601 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     6661 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     6721 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6781 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6841 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6901 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6961 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     7021 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     7081 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     7141 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     7201 gcctctt
//
