LOCUS       sequence-445453-        6945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 based UAS modified with a CCR5TC binding site upstream of it, 
            driving PSD95-FingR fused to GFP, CCR5TC zinc finger, and KRAB(A) 
            repressor domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6945)
  TITLE     Bonkowsky lab FingR plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6945)
  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: 
            "Unpublished"
COMMENT     Sequence Label: zcUAS:PSD95.FingR-GFP-ZFC(CCR5TC)-KRAB(A)
FEATURES             Location/Qualifiers
     source          1..6945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1117
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     1119..1138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1119..1137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1190..1294)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    1345..1356
                     /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)"
     primer_bind     complement(1923..1939)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(1976..1994)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    1994..2018
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2292..2315
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2340..2342,2343..3050)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2340..2342,2343..3050)
                     /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(2382..2403)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2643..2662)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2990..3011
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3096..3437
                     /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             3486..3611
                     /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"
     protein_bind    complement(3619..3643)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3645..3779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(3842..3862)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(3870..3886)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3902..4036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(4475..4662,4663..4674)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4708..4724)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(4761..4779)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4800..4816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4813..4835)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4824..4840
                     /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(4848..4854,4855..4872,4873..4878))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4893..4914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5031..5048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5202..5790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5282..5301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5961..6752,6753..6821))
                     /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     6584..6603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6822..6926)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 atgctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttggta atacgactca ctatagggcg
     1141 aattgggtac caagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     1201 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     1261 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     1321 ggtcgacatt gattattgac tagagtcatc ctcatcgtcg acattgatta ttgactagtt
     1381 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1441 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1501 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1561 tggactattt acggtaagct taggcctcca aggcggagta ctgtcctccg ggctggcgga
     1621 gtactgtcct ccggcaaggt cggagtactg tcctccgaca ctagaggtcg gagtactgtc
     1681 ctccgacgca aggcggagta ctgtcctccg ggctgcggag tactgtcctc cggcaaggtc
     1741 ggagtactgt cctccgacac tagaggtcgg agtactgtcc tccgacgcaa ggcggagtac
     1801 tgtcctccgg gctggcggag tactgtcctc cggcaagggt cgactctaga gggtatataa
     1861 tggatcccat cgcgtctcag cctcactttg agctcctcca cacgaattcc tgcagcccgg
     1921 gggatccact agttctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt
     1981 agtgagggtt aattcaagtt tgtacaaaaa agcaggctac catgctcgaa gtcaaggaag
     2041 catcaccaac cagcatccag atcagctggg tgctccactt gcgccacgtt cgctactacc
     2101 gcatcaccta cggtgaaact ggtggcaata gccctgtcca ggaattcacc gtgcctggca
     2161 gcaagtccac tgctaccatc agcggcctga aacctggtgt cgactatacc atcacggtgt
     2221 acgccgtcac gatcttcagc gcctaccgct ccgcctggcc gccgatctcc atcaactacc
     2281 gcaccggaac cgattacaag gatgacgacg ataagggtag cggctccagt agatctgggg
     2341 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2401 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2461 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2521 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2581 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2641 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2701 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2761 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2821 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2881 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2941 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3001 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac tctaggggag
     3061 ctggcgctgg agcgggtgca ggggctggct ctagattcca gtgccggatc tgcatgcgga
     3121 acttcagcga ccggtccaac ctgagcaggc acatcagaac ccacaccgga gaaaagccct
     3181 tcgcctgcga catttgcggc cggaagttcg ccatcagcag caacctgaac agccacacca
     3241 agatccacac tggcagccag aaacctttcc agtgcagaat ttgtatgaga aactttagca
     3301 gaagcgacaa cctggccaga cacatccgga cacatactgg tgaaaaacct tttgcctgtg
     3361 atatctgtgg cagaaagttt gccacctccg gcaatctgac ccggcacaca aagattcacc
     3421 tgcggggcag ccagctatcg attgtcgacg ctcctgaaca acgtgaaggt gcttctcaag
     3481 tttctgtttc tgttactttt gaagatgttg ctgttctttt tactcgtgat gaatggaaaa
     3541 aacttgatct ttctcaacgt tctctttatc gtgaagttat gcttgaaaat tattctaatc
     3601 ttgcttctat ggcttaatac ccagctttct tgtacaaagt gggggatcca gacatgataa
     3661 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3721 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3781 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     3841 ccaactttat tatacatagt tgataattca ctggccgtcg ttttacggta ccatcgatga
     3901 tgatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3961 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4021 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4081 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     4141 tcctctagat cagatctgcg aagatacggc cacgggtgct cttgatcctg tggctgattt
     4201 tggactgtgc tgctcgcagc tgctgatgaa tcacatactt cctccatttt cttccactga
     4261 ttgactgtta taatttccct aatttccagg tcaaggtgct gtgcattgtg gtaatagatg
     4321 tgacatgacg tcacttccaa aggaccaatg aacatgtctg accaatttca tataatgtga
     4381 aaacgatttt cataggcaga ataaataaca tttaaattaa actgggcatc agcgcaattc
     4441 aattggtttg gtaatagcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt
     4501 tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat
     4561 taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa
     4621 ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt
     4681 gaccccctac cttcagcaag cccagcagat ccactagttc tagagcggcc gccaccgcgg
     4741 tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     4801 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4861 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4921 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4981 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5041 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5101 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5161 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5221 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5281 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5341 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5401 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5461 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5521 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5581 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5641 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5701 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5761 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5821 gacgctcagt ggaacgaaaa ctcacgttaa ggcattttgg tcatgagatt atcaaaaagg
     5881 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5941 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6001 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6061 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6121 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6181 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6241 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6301 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6361 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6421 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6481 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6541 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6601 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6661 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6721 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6781 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6841 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6901 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtg
//
