LOCUS       sequence-405268-        6449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of rsEGFP2-E in mamalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6449)
  AUTHORS   Qian Y, Celiker OT, Wang Z, Guner-Ataman B, Boyden ES
  TITLE     Temporally multiplexed imaging of dynamic signaling networks in 
            living cells.
  JOURNAL   Cell. 2023 Dec 7;186(25):5656-5672.e21. doi: 
            10.1016/j.cell.2023.11.010. Epub 2023 Nov 28.
  PUBMED    38029746
REFERENCE   2  (bases 1 to 6449)
  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.cell.2023.11.010"; journalName: "Cell"; date: "2023-12-7-
            7"; volume: "186"; issue: "25"; pages: "5656-5672.e21"
COMMENT     Sequence Label: pcDuex2-rsEGFP2-E
FEATURES             Location/Qualifiers
     source          1..6449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             944..1660
                     /codon_start=1
                     /product="reversibly and rapidly switchable enhanced green 
                     fluorescent protein (Grotjohann et al., 2012)"
                     /label=rsEGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLAYGVLCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     SNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(989..1010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1250..1269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1597..1618
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1667..2005
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVS"
     primer_bind     complement(2040..2057)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2046..2270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2316..2744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2403..2422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2613..2634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2753..2773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2758..3087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2938..3073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3000..3019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3154..3948
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3208..3227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3818..3837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4122..4243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4159..4178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4213..4232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4292..4308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4292..4308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4305..4327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4316..4332
                     /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(4340..4346,4347..4364,4365..4370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4385..4406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4523..4540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4694..5282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4774..4793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5453..6244,6245..6313))
                     /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     6076..6095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6314..6418)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gtccggattc aattaatcat cggctcgtat aatatacccg tttttttggg
      901 ctagaaataa ttttgtctaa ctttaagaag gagatatctc gagatggtga gcaagggcga
      961 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1021 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1081 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctggc
     1141 ctacggcgtg ctgtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1201 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1261 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1321 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1381 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca agtctaactt
     1441 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1501 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1561 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1621 cgccgccggg atcactctcg gcatggacga gctgtacaag ctgcaagttc aactggtgga
     1681 gtcaggtggt gcacttgttc agccgggcgg tagtttgcga ctttcatgcg cggcgtcagg
     1741 tttccctgta aaccgataca gtatgaggtg gtatagacaa gcgcccggca aagaacgaga
     1801 atgggttgca ggaatgagta gtgcaggaga tcgaagttcc tacgaagaca gcgttaaagg
     1861 gcgcttcact atcagtcgag atgatgcacg gaacaccgtg tatctgcaaa tgaacagcct
     1921 taaacctgaa gatacggccg tatattattg taacgtcaac gtcggatttg aatactgggg
     1981 acagggtacg caagtcacgg tatcttaatc tagagggccc gtttaaaccc gctgatcagc
     2041 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2101 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2161 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2221 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2281 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2761 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2821 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2881 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2941 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3001 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3061 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3121 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3601 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3661 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3841 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4141 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4201 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4261 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4321 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4381 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4441 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4501 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4561 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4621 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4681 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4741 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4801 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4861 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4921 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4981 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5041 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5101 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5161 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5221 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5281 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5341 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5401 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5461 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5521 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5581 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5641 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5701 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5761 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5821 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5881 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5941 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6001 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6061 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6121 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6181 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6241 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6301 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6361 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6421 catttccccg aaaagtgcca cctgacgtc
//
