LOCUS       sequence-202869-        7590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP in sensory neurons (retinal ganglion cells, 
            trigeminal, Rohon Beard neurons and dorsal root ganglia) for up to 2
            weeks.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7590)
  TITLE     Rieger lab plasmids
REFERENCE   2  (bases 1 to 7590)
  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"
COMMENT     Sequence Label: pDest-Tol2 (Chien)-Tol2-Isl2b:GFP:polyA
FEATURES             Location/Qualifiers
     source          1..7590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(771..789)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    794..928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(991..1011)
                     /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(1019..1035)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1051..1185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(1624..1811,1812..1823)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(1857..1873)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1857..1873)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1910..1930)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1910..1928)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1949..1965)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1962..1984)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1973..1989
                     /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(1997..2003,2004..2021,2022..2027))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2042..2063
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2180..2197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2351..2939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2431..2450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3110..3901,3902..3970))
                     /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     3733..3752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3971..4075)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4238..4259)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4608..4619,4620..4782)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     5155..5171
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5191..5211
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    6073..6097
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6181..6621
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     polyA_signal    6894..7028
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(7562..7586)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      join(7585..7590,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc
      721 ggtggaagat ctgggaattc aaggcctctc gagcctctag attctgcagc cctatagtga
      781 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
      841 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      901 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
      961 ggttcagggg gaggtgtggg aggttttttc caactttatt atacatagtt gataattcac
     1021 tggccgtcgt tttacggtac catcgatgat gatccagaca tgataagata cattgatgag
     1081 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1141 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1201 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     1261 ctctacaaat gtggtatggc tgattatgat cctctagatc agatctgcga agatacggcc
     1321 acgggtgctc ttgatcctgt ggctgatttt ggactgtgct gctcgcagct gctgatgaat
     1381 cacatacttc ctccattttc ttccactgat tgactgttat aatttcccta atttccaggt
     1441 caaggtgctg tgcattgtgg taatagatgt gacatgacgt cacttccaaa ggaccaatga
     1501 acatgtctga ccaatttcat ataatgtgaa aacgattttc ataggcagaa taaataacat
     1561 ttaaattaaa ctgggcatca gcgcaattca attggtttgg taatagcaag ggaaaataga
     1621 atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa
     1681 aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt
     1741 gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat
     1801 tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc
     1861 cactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     1921 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1981 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2041 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2101 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2161 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2221 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2281 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2341 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2401 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2461 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2521 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2581 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2641 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2701 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2761 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2821 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2881 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2941 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3001 gcattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3061 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3121 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3181 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3241 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3301 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3361 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3421 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3481 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3541 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3601 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3661 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3721 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3781 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3841 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3901 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3961 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4021 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4081 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     4141 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4201 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4261 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4321 acgctggcta agaactcatc agcctccccg gtccatctac ccacgtacca atgcaccaat
     4381 tggccacaat gacggctact acatggtgcc attccttcct ctttatagga atggagacta
     4441 cctcctgtcc aacaatgctc ttggatacga gtacgcctac ctgttggacc caggtcattg
     4501 cacaacacca gaaatgccct ctgatctgca aaagacgtga atatctgttc agacacccat
     4561 atccactctg ttccacacag gtcagaggtt tgtccaggag ttcttgacag aggtgtaaaa
     4621 agtactcaaa aattttactc aagtgaaagt acaagtactt agggaaaatt ttactcaatt
     4681 aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa gtaaaaaagt actccattaa
     4741 aattgtactt gagtattaag gaagtaaaag taaaagcaag aaagaaaact agagattctt
     4801 gtttaagctt ttaatctcaa aaaacattaa atgaaatgca tacaaggttt tatcctgctt
     4861 tagaactgtt tgtatttaat tatcaaacta taagacagac aatctaatgc cagtacacgc
     4921 tactcaaagt tgtaaaacct cagatttaac ttcagtagaa gctgattctc aaaattgtta
     4981 gtgtcaagcc tagctctttt ggggctgaaa agcaatcctg cagtgctgaa aagcctctca
     5041 caggcagccg atgcgggaag aggtgtatta gtcttgatag agaggctgca aatagcagga
     5101 aacgtgagca gagactccct ggtgtctgaa acacaggcca gatgggccct cgagcaggaa
     5161 acagctatga ccatgattac gccaagctat caactttgta tagaaaagtt ggtaacagga
     5221 tgtgacacgt cgtctgcttt aatcagtgtt tgagagggca cctctacaca ctgaatgcca
     5281 agcaaagaaa gtcagaaaat ctaaataata gcaaacatac acacacacac acatacacaa
     5341 atatatatat caactcttca ttcagttatt tacaaaaaaa aacagaatat ctgaactagc
     5401 cagatgaaaa agagagcgag aaaggagaaa agaaagagag agagagataa aatgaaagag
     5461 acagacagaa acagacagag ggagagggag tcagtctctt gtgacctaca tgaaagcaac
     5521 gtgcctcgct actcctggct aaatgcactc tcatattaat attttaaaac gccgtaatgt
     5581 gacggtgaaa ccctcggagg acagatacat caatttgcag gggcctcggc ttcaggaaag
     5641 agaaacaggg ataatttgcc attaggagaa attgttcatt atgggcagct tcataggaac
     5701 acataaacca agtcacctct ctcacgtgag ctgcaaatcc acacttttgc gtgaatggag
     5761 acgttgcccg agagctttaa tgctcctggg tcattagtac aaaaaggttt gttgtgctgt
     5821 taaatctgct gcatgttgcg gtgatcaatc aacatgaaag gggcttccca ctaagaccgg
     5881 atcactgtaa ataaatcaat taggccgcga gagtcaagct tttatcagaa ttgttgctac
     5941 acacacacac acacacacac aatcggccta tagattcagt gctgaacagt ccggttcaga
     6001 ttcagctcaa gataaataag acatctgagt ttgctgatca tcgttcagcc agtaaatgac
     6061 accagcagca ggccaagttt gtacaaaaaa gcaggctcaa tcgccctttc tagagggtat
     6121 ataatggatc ccatcgcgtc tcagcctcac tttgagctcc tccacacgaa ttctttggcc
     6181 atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag
     6241 tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac
     6301 tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg
     6361 ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt
     6421 ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat
     6481 aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta
     6541 acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt
     6601 caaagacagt tgactgtatc gtcgaggtcg accccgggaa ttcagatctc tcgagccgcc
     6661 cccccgaccg atgtcagcct gggggacgag ctccacttag acggcgagga cgtggcgatg
     6721 gcgcatgccg acgcgctaga cgatttcgat ctggacatgt tgggggacgg ggattccccg
     6781 ggtccgggat ttacccccca cgactccgcc ccctacggcg ctctggatat gtagcggatc
     6841 tactagtgcg gcccctctcg agcctctaga actatagtga gtcgtattac gtagatccag
     6901 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     6961 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     7021 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     7081 aggtttttta attcgcggcc atcaagctta ggcctccaag gcggagtact gtcctccggg
     7141 ctggcggagt actgtcctcc ggcaaggtcg gagtactgtc ctccgacact agaggtcgga
     7201 gtactgtcct ccgacgcaag gcggagtact gtcctccggg ctgcggagta ctgtcctccg
     7261 gcaaggtcgg agtactgtcc tccgacacta gaggtcggag tactgtcctc cgacgcaagg
     7321 tcggagtact gtcctccgac actagaggtc ggagtactgt cctccgacgc aaggtcggag
     7381 tactgtcctc cgacactaga ggtcggagta ctgtcctccg acgcaaggcg gagtactgtc
     7441 ctccgggctg gcggagtact gtcctccggc aagggtcgac tctagagggt atataatgga
     7501 tcccatcgcg tctcagcctc actttgagct cctccacacg aattccctcg acctcgaaga
     7561 cacccagctt tcttgtacaa agtggccacc
//
