LOCUS       sequence-250418-        7193 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 7193)
  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 7193)
  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..7193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    145..156
                     /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        168..1346
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          399..1337
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1294..1314
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1363..1382
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1363..1381
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1444..1477)
                     /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(1528..1561)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1576..1701)
                     /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(1750..2091)
                     /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(2137..2844,2845..2847))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             complement(join(2137..2844,2845..2847))
                     /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(2176..2197)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2525..2544
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2784..2805
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2872..2895)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3178..3211
                     /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    3262..3295
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3320..3908
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3373..3393)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3791..3802)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3940..4416
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4139..4158)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4456..4596
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4456..4585
                     /label=AAV2 ITR
     rep_origin      4671..5126
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4758..4777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4968..4989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5143..5162)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5262..5284
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5322..5340)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5408..5512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5513..5581,5582..6373)
                     /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(5731..5750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6544..7132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7033..7052
                     /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 gcgtgtcatc ctcatcgaat tccgtgaggc tccggtgccc
      181 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
      241 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
      301 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
      361 acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc
      421 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc
      481 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc
      541 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg
      601 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc
      661 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
      721 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
      781 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
      841 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
      901 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
      961 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
     1021 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
     1081 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct
     1141 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     1201 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     1261 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     1321 tttttcttcc atttcaggtg tcgtgaggaa ttagcttggt actaatacga ctcactatag
     1381 ggagacccaa gctggctagg taagcttggt accgagctcg gatcctctag agtcgactcc
     1441 ggaataactt cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag
     1501 ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta
     1561 tggcgcgcct taagccatag aagcaagatt agaataattt tcaagcataa cttcacgata
     1621 aagagaacgt tgagaaagat caagtttttt ccattcatca cgagtaaaaa gaacagcaac
     1681 atcttcaaaa gtaacagaaa cagaaacttg agaagcacct tcacgttgtt caggagcgtc
     1741 gacaatcgat agctggctgc cccgcaggtg aatctttgtg tgccgggtca gattgccgga
     1801 ggtggcaaac tttctgccac agatatcaca ggcaaaaggt ttttcaccag tatgtgtccg
     1861 gatgtgtctg gccaggttgt cgcttctgct aaagtttctc atacaaattc tgcactggaa
     1921 aggtttctgg ctgccagtgt ggatcttggt gtggctgttc aggttgctgc tgatggcgaa
     1981 cttccggccg caaatgtcgc aggcgaaggg cttttctccg gtgtgggttc tgatgtgcct
     2041 gctcaggttg gaccggtcgc tgaagttccg catgcagatc cggcactgga atctagagcc
     2101 agcccctgca cccgctccag cgccagctcc cctagagtac agctcgtcca tgccgagagt
     2161 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     2221 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     2281 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     2341 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     2401 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     2461 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     2521 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     2581 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     2641 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     2701 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     2761 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     2821 ggtgaacagc tcctcgccct tgctcacccc agatctactg gagccgctac ccttatcgtc
     2881 gtcatccttg taatcggttc cggtgcggta gttgatggag atcggcggcc aggcggagcg
     2941 gtaggcgctg aagatcgtga cggcgtacac cgtgatggta tagtcgacac caggtttcag
     3001 gccgctgatg gtagcagtgg acttgctgcc aggcacggtg aattcctgga cagggctatt
     3061 gccaccagtt tcaccgtagg tgatgcggta gtagcgaacg tggcgcaagt ggagcaccca
     3121 gctgatctgg atgctggttg gtgatgcttc cttgacttcg agcatggtgg cgctagcata
     3181 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta
     3241 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt
     3301 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3361 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3421 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3481 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3541 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3601 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3661 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3721 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3781 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3841 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3901 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     3961 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4021 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4081 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4141 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4201 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4261 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4321 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4381 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4441 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4501 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4561 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4621 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4681 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4741 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4801 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4861 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4921 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4981 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5041 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5101 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5161 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5221 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5281 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5341 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6661 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6721 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6781 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6841 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6901 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6961 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7021 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7081 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7141 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
