LOCUS       sequence-241728-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Labeling of excitatory synapses (green fluorescence) in 
            cre-expressing neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  AUTHORS   Bensussen S, Shankar S, Ching KH, Zemel D, Ta TL, Mount RA, Shroff 
            SN, Gritton HJ, Fabris P, Vanbenschoten H, Beck C, Man HY, Han X
  TITLE     A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.
  JOURNAL   iScience. 2020 Jun 30;23(7):101330. doi: 10.1016/j.isci.2020.101330.
  PUBMED    32674057
REFERENCE   2  (bases 1 to 7204)
  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: "iScience.";
            date: "2020-06-30"; volume: "23(7):101330. doi"; pages: " 
            10.1016/j.isci.2020.101330"
COMMENT     Sequence Label: AAV-EF1A-DIO-PSD95.FingR-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    156..167
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     promoter        179..1357
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          410..1348
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1305..1325
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1374..1393
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1374..1392
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1455..1488)
                     /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(1539..1572)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1587..1712)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the rat zinc finger protein Kid-1 
                     (Witzgall et al., 1994)"
                     /label=KRAB-A
                     /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM"
     CDS             complement(1761..2102)
                     /codon_start=1
                     /product="zinc-finger domain from the CCR5-224 (+) 
                     zinc-finger nuclease that recognizes the left half-site 
                     target sequence in the human CCR5 gene (Perez et al., 2008;
                     Gross et al., 2013)"
                     /label=CCR5TC
                     /translation="FQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLN
                     SHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRH
                     TKIHLRGSQL"
     CDS             complement(join(2148..2855,2856..2858))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             complement(join(2148..2855,2856..2858))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(2187..2208)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2536..2555
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2795..2816
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2883..2906)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3189..3222
                     /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    3273..3306
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3331..3919
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3384..3404)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3802..3813)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3951..4427
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4150..4169)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4467..4607
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4467..4596
                     /label=AAV2 ITR
     rep_origin      4682..5137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4769..4788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4979..5000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5154..5173)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5273..5295
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5333..5351)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5419..5523
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5524..5592,5593..6384)
                     /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(5742..5761)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6555..7143
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7044..7063
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgtcat cctcatcgaa ttccgtgagg
      181 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      241 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      301 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      361 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
      421 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
      481 ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
      541 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc
      601 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
      661 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
      721 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
      781 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
      841 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
      901 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
      961 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
     1021 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     1081 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     1141 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     1201 gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     1261 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     1321 ggttcaaagt ttttttcttc catttcaggt gtcgtgagga attagcttgg tactaatacg
     1381 actcactata gggagaccca agctggctag gtaagcttgg taccgagctc ggatcctcta
     1441 gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag
     1501 ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt
     1561 atacgaagtt atggcgcgcc ttaagccata gaagcaagat tagaataatt ttcaagcata
     1621 acttcacgat aaagagaacg ttgagaaaga tcaagttttt tccattcatc acgagtaaaa
     1681 agaacagcaa catcttcaaa agtaacagaa acagaaactt gagaagcacc ttcacgttgt
     1741 tcaggagcgt cgacaatcga tagctggctg ccccgcaggt gaatctttgt gtgccgggtc
     1801 agattgccgg aggtggcaaa ctttctgcca cagatatcac aggcaaaagg tttttcacca
     1861 gtatgtgtcc ggatgtgtct ggccaggttg tcgcttctgc taaagtttct catacaaatt
     1921 ctgcactgga aaggtttctg gctgccagtg tggatcttgg tgtggctgtt caggttgctg
     1981 ctgatggcga acttccggcc gcaaatgtcg caggcgaagg gcttttctcc ggtgtgggtt
     2041 ctgatgtgcc tgctcaggtt ggaccggtcg ctgaagttcc gcatgcagat ccggcactgg
     2101 aatctagagc cagcccctgc acccgctcca gcgccagctc ccctagagta cagctcgtcc
     2161 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     2221 tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc
     2281 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     2341 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     2401 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     2461 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     2521 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     2581 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     2641 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     2701 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     2761 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     2821 ggcaccaccc cggtgaacag ctcctcgccc ttgctcaccc cagatctact ggagccgcta
     2881 cccttatcgt cgtcatcctt gtaatcggtt ccggtgcggt agttgatgga gatcggcggc
     2941 caggcggagc ggtaggcgct gaagatcgtg acggcgtaca ccgtgatggt atagtcgaca
     3001 ccaggtttca ggccgctgat ggtagcagtg gacttgctgc caggcacggt gaattcctgg
     3061 acagggctat tgccaccagt ttcaccgtag gtgatgcggt agtagcgaac gtggcgcaag
     3121 tggagcaccc agctgatctg gatgctggtt ggtgatgctt ccttgacttc gagcatggtg
     3181 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     3241 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     3301 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3361 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3421 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3481 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3541 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3601 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3661 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3721 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     3781 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3841 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3901 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     3961 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     4021 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     4081 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     4141 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     4201 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     4261 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     4321 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     4381 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     4441 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     4501 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4561 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4621 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4681 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4741 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4801 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4861 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4921 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4981 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     5041 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5101 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5161 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5221 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5281 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5341 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5401 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5461 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5521 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5581 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5641 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5701 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5761 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5821 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5881 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5941 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6001 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6061 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6121 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6181 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6241 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6301 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6361 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6421 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6481 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6541 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6601 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6661 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6721 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6781 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6841 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6901 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6961 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7021 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7081 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7141 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7201 atgt
//
