LOCUS       sequence-65197-e        7497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7497)
  AUTHORS   Gross GG, Junge JA, Mora RJ, Kwon HB, Olson CA, Takahashi TT, Liman 
            ER, Ellis-Davies GC, McGee AW, Sabatini BL, Roberts RW, Arnold DB
  TITLE     Recombinant probes for visualizing endogenous synaptic proteins in 
            living neurons.
  JOURNAL   Neuron. 2013 Jun 19;78(6):971-85. doi: 10.1016/j.neuron.2013.04.017.
  PUBMED    23791193
REFERENCE   2  (bases 1 to 7497)
  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"; doi: 
            "10.1016/j.neuron.2013.04"; journalName: "Neuron"; date: 
            "2013-06-19- 19"; volume: "78"; issue: "6"; pages: "971-85"
COMMENT     Sequence Label: pCAG_GPHN.FingR-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    24..35
                     /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)"
     enhancer        39..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        421..696
                     /label=chicken beta-actin promoter
     intron          697..1708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1632..1654
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1716..1735
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2056..2079
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2101..2103,2104..2811)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2101..2103,2104..2811)
                     /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(2143..2164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2404..2423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2751..2772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2857..3198
                     /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             3247..3372
                     /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"
     intron          3421..3553
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3536..3555
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3562..4150
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3615..3635)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4033..4044)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4177..4194)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4183..4294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4586..4641
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4640..4659)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5000..5016)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5013..5035)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5024..5040
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5048..5054,5055..5072,5073..5078))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5093..5114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5173..5368
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5219..5354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5374..5508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5411..5430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5465..5484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5576..5593)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5747..6335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5827..5846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6506..7297,7298..7366))
                     /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     7129..7148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7367..7471)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agagtcatcc tcatcgtcga cattgattat tgactagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggg
      421 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
      481 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
      541 cgcgcgccag gcggggcggg gcgggggcga gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtt gccttcgccc
      721 cgtgccccgc tccgcgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
      841 tgacggctcg tttcttttct gtggctgcgt gaaagcctta aagggctccg ggagggccct
      901 ttgtgcgggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
      961 gtgcggcccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1021 ctccgcgtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc gggggggctg
     1081 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     1141 cgcggcggtc gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc
     1201 cggcttcggg tgcggggctc cgtgcggggc gtggcgcggg gctcgccgtg ccgggcgggg
     1261 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     1321 gggaggggcg cggcggcccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca
     1381 ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc
     1441 ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg
     1501 cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc
     1561 ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg
     1621 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     1681 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc
     1741 tcatcatttt ggcaaagaat tcggtaccgc gggcccggga ttacaatgct cgaagtcaag
     1801 gaagcatcac caaccagcat ccagatcagc tggggcaagt acaaggtcat ggttcgctac
     1861 taccgcatca cctacggtga aactggtggc aatagccctg tccaggaatt caccgtgcct
     1921 ggcagcaagt ccactgctac catcagcagc ctgaaacctg gtgtcgacta taccatcacg
     1981 gtgtacgccg tcacgatcga ccactggaac taccaggacc cgatcccgat ctccatcaac
     2041 taccgcaccg gatccgatta caaggatgac gacgataagg gtagcggctc cagatctggg
     2101 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2161 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2221 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2281 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2341 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2401 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2461 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2521 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2581 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2641 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2701 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2761 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta ctctagggga
     2821 gctggcgctg gagcgggtgc aggggctggc tctagattcc agtgccggat ctgcatgcgg
     2881 aacttcagcg accggtccaa cctgagcagg cacatcagaa cccacaccgg agaaaagccc
     2941 ttcgcctgcg acatttgcgg ccggaagttc gccatcagca gcaacctgaa cagccacacc
     3001 aagatccaca ctggcagcca gaaacctttc cagtgcagaa tttgtatgag aaactttagc
     3061 agaagcgaca acctggccag acacatccgg acacatactg gtgaaaaacc ttttgcctgt
     3121 gatatctgtg gcagaaagtt tgccacctcc ggcaatctga cccggcacac aaagattcac
     3181 ctgcggggca gccagctatc gattgtcgac gctcctgaac aacgtgaagg tgcttctcaa
     3241 gtttctgttt ctgttacttt tgaagatgtt gctgttcttt ttactcgtga tgaatggaaa
     3301 aaacttgatc tttctcaacg ttctctttat cgtgaagtta tgcttgaaaa ttattctaat
     3361 cttgcttcta tggcttaaac gcgttgtaca agtaaagcgg ccccggactc gaggccgcag
     3421 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga
     3481 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc
     3541 tttctctcca caggtgtcga caatcaacct ctggattaca aaatttgtga aagattgact
     3601 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3661 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3721 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3781 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3841 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3901 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     3961 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4021 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4081 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4141 gcctccccgc ctggaattcg agctcggtac gatcagcctc gactgtgcct tctagttgcc
     4201 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4261 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4321 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4381 cagcttttgt tccctttagg gttaattgcg cgcttggcgc taatcatggt catagctgtt
     4441 tccactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     4501 gctcacaaat accactgaga tcgttttccc tctgccaaaa attatgggga catcatgaag
     4561 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     4621 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4681 cagaatgagt atttggttta gagtttggca acatatgcca tatgctggct gccatgaaca
     4741 aaggtggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     4801 ccatagaaaa gccttgactt gaggttagat ttttttttat attttgtttg tgttattttt
     4861 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     4921 ctgactactc ccagtcatag ctgtccctct tctcttatga agatccctcg acctgcagcc
     4981 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5041 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5101 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5161 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5221 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5281 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5341 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     5401 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5461 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     5521 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5581 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5641 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5701 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5761 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5821 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5881 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5941 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6001 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6061 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6121 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6181 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6241 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6301 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6361 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6421 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6481 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6541 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6601 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6661 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6721 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6781 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6841 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6901 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6961 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7021 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7081 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7141 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7201 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7261 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7321 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7381 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7441 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
