LOCUS       sequence-177097-        4854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP reporter vector for iTango system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4854)
  AUTHORS   Lee D, Creed M, Jung K, Stefanelli T, Wendler DJ, Oh WC, Mignocchi 
            NL, Luscher C, Kwon HB
  TITLE     Temporally precise labeling and control of neuromodulatory circuits 
            in the mammalian brain.
  JOURNAL   Nat Methods. 2017 May;14(5):495-503. doi: 10.1038/nmeth.4234. Epub 
            2017 Apr 3.
  PUBMED    28369042
REFERENCE   2  (bases 1 to 4854)
  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: 
            "10.1038/nmeth.4234"; journalName: "Nat Methods"; date: "2017-05"; 
            volume: "14"; issue: "5"; pages: "495-503"
COMMENT     Sequence Label: pTRE-EGFP
FEATURES             Location/Qualifiers
     source          1..4854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(21..39,40..62,63..81,82..104,105..123,124..146,
                     147..165,166..188,189..207,208..230,231..249,250..272,
                     273..291)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(63..81,82..104,105..123,124..146,147..165,166..188,
                     189..207,208..230,231..249,250..272,273..291,292..314,
                     315..333)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(105..123,124..146,147..165,166..188,189..207,208..230,
                     231..249,250..272,273..291,292..314,315..333,334..356,
                     357..375)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(147..165,166..188,189..207,208..230,231..249,250..272,
                     273..291,292..314,315..333,334..356,357..375,376..398,
                     399..417)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(189..207,208..230,231..249,250..272,273..291,292..314,
                     315..333,334..356,357..375,376..398,399..417,418..440,
                     441..459)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(231..249,250..272,273..291,292..314,315..333,334..356,
                     357..375,376..398,399..417,418..440,441..459,460..482,
                     483..501)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    join(273..291,292..314,315..333,334..356,357..375,376..398,
                     399..417,418..440,441..459,460..482,483..501,502..524,
                     525..543)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     primer_bind     989..1013
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1129..1131,1132..1134,1135..1848)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1129..1131,1132..1134,1135..1845)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1174..1195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1782..1803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1857..1876)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1858..1876)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1881..2015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1905..1924)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1959..1978
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2128..2148)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2128..2146)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2167..2183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2167..2183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2180..2202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2191..2207
                     /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(2215..2221,2222..2239,2240..2245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2260..2281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2398..2415)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2568..3156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2648..2667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3327..4118,4119..4187))
                     /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     3950..3969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4188..4292)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4318..4773)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4455..4476)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4667..4686
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccctcgatcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
       61 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag
      121 agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag
      181 tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag
      241 tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa
      301 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
      361 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
      421 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
      481 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag
      541 agaaaagtga aagtcgagct cggtacgcta tggcatgcat gtgtcgcgac ctgcaggccc
      601 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      661 tgacctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
      721 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      781 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
      841 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
      901 acgccatcag cgacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
      961 acttcaagag ctagccctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     1021 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg
     1081 gccccggtac cgaattcaag gcctctcgag cctctagaag gtggcggaat ggtgagcaag
     1141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1261 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1321 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1381 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1561 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1621 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1741 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1801 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaggc tagcctccct
     1861 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     1921 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1981 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     2041 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcgtccgcg gcgccaatgc
     2101 attgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     2161 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2221 acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2281 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2341 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2401 gctcactgac tcgctgcgct cggtcgttcg gctgcgggag cggtatcagc tcactcaaag
     2461 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2521 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2581 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2641 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2761 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2821 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2881 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2941 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3001 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3061 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3121 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3181 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3241 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3301 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3361 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3421 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3481 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3541 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3601 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3661 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3721 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3781 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3841 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3901 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3961 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4021 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4081 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4141 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4201 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4261 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     4321 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4381 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4441 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4501 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4561 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4621 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4681 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4741 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     4801 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacg atat
//
