LOCUS       sequence-252008-        6553 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Global labeling of inhibitory synapses (green fluorescence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6553)
  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 6553)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Gephyrin.FingR-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..6553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    74..85
                     /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        97..1275
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          328..1266
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1223..1243
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1292..1311
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1292..1310
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1634..1657
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(1682..1684,1685..2392)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(1682..1684,1685..2392)
                     /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(1724..1745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1985..2004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2332..2353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2438..2779
                     /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             2828..2953
                     /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"
     misc_feature    2972..3560
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3025..3045)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3443..3454)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3563..3579)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3564..3580)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3609..3730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3620..3639)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3674..3693
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4109..4697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4189..4208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4868..5659,5660..5728))
                     /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     5491..5510
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5729..5833)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5859..6314)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5996..6017)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6208..6227
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6386..6515
                     /label=AAV2 ITR
ORIGIN
        1 tagccatgct ctaggaagat ctggacagca gagatccagt ttggatcgat aagcttgata
       61 tcgaattacg cgtgtcatcc tcatcgaatt ccgtgaggct ccggtgcccg tcagtgggca
      121 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
      181 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      241 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      301 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
      361 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
      421 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
      481 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
      541 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
      601 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
      661 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
      721 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
      781 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
      841 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
      901 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
      961 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     1021 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     1081 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     1141 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     1201 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     1261 tttcaggtgt cgtgaggaat tagcttggta ctaatacgac tcactatagg gagacccaag
     1321 ctggctaggt aagcttggta ccgagctcgg atccgccgcc accatgctcg aagtcaagga
     1381 agcatcacca accagcatcc agatcagctg gggcaagtac aaggtcatgg ttcgctacta
     1441 ccgcatcacc tacggtgaaa ctggtggcaa tagccctgtc caggaattca ccgtgcctgg
     1501 cagcaagtcc actgctacca tcagcagcct gaaacctggt gtcgactata ccatcacggt
     1561 gtacgccgtc acgatcgacc actggaacta ccaggacccg atcccgatct ccatcaacta
     1621 ccgcaccgga tccgattaca aggatgacga cgataagggt agcggctcca gtagatctgg
     1681 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1741 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1801 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1861 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1921 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1981 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2041 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2101 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2161 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2221 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2281 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2341 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt actctagggg
     2401 agctggcgct ggagcgggtg caggggctgg ctctagattc cagtgccgga tctgcatgcg
     2461 gaacttcagc gaccggtcca acctgagcag gcacatcaga acccacaccg gagaaaagcc
     2521 cttcgcctgc gacatttgcg gccggaagtt cgccatcagc agcaacctga acagccacac
     2581 caagatccac actggcagcc agaaaccttt ccagtgcaga atttgtatga gaaactttag
     2641 cagaagcgac aacctggcca gacacatccg gacacatact ggtgaaaaac cttttgcctg
     2701 tgatatctgt ggcagaaagt ttgccacctc cggcaatctg acccggcaca caaagattca
     2761 cctgcggggc agccagctat cgattgtcga cgctcctgaa caacgtgaag gtgcttctca
     2821 agtttctgtt tctgttactt ttgaagatgt tgctgttctt tttactcgtg atgaatggaa
     2881 aaaacttgat ctttctcaac gttctcttta tcgtgaagtt atgcttgaaa attattctaa
     2941 tcttgcttct atggcttaaa agcttatcga taatcaacct ctggattaca aaatttgtga
     3001 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3061 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3121 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3181 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3241 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3301 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3361 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3421 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3481 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3541 cctttgggcc gcctccccgc atcgataccg tcgacctcga cccgggcggc cgcttcgagc
     3601 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3661 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3721 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     3781 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatcttcc
     3841 tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc
     3901 tagtgatgga gttggccact ccctctctgy scgctysctc gctcactgac tcgctgcgct
     3961 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4021 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4081 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4141 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4201 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4261 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4321 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4381 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4441 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4501 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4561 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4621 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4681 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4741 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4801 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4861 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4921 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4981 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5041 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5101 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5161 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5221 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5281 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5341 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5401 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5461 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5521 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5581 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5641 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5701 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5761 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5821 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     5881 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5941 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6001 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6061 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6121 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6181 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6241 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6301 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     6361 gcgggcctct tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc
     6421 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     6481 agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg
     6541 ctacttatct acg
//
