LOCUS       sequence-444999-        7119 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 7119)
  AUTHORS   Fritz RD, Letzelter M, Reimann A, Martin K, Fusco L, Ritsma L, 
            Ponsioen B, Fluri E, Schulte-Merker S, van Rheenen J, Pertz O
  TITLE     A Versatile Toolkit to Produce Sensitive FRET Biosensors to 
            Visualize Signaling in Time and Space.
  JOURNAL   Sci Signal. 2013 Jul 23;6(285):rs12. doi: 10.1126/scisignal.2004135.
  PUBMED    23882122
REFERENCE   2  (bases 1 to 7119)
  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: "Sci 
            Signal."; date: "2013-07-23"; volume: "6(285):rs12. doi"; pages: " 
            10.1126/scisignal.2004135"
COMMENT     Sequence Label: EKAR2G_design1_mTFP_159_Venus_173
FEATURES             Location/Qualifiers
     source          1..7119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        22..325
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        327..529
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     480..500
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     732..754
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     816..835
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     877..896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        877..895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    896..920
                     /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        936..1045
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             1066..1083
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1093..1122
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2443..3177
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     2580..2601
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2704..2725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2965..2984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3247..3270
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3360..3379)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3425..3480
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3479..3498)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3568..3615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3628..4333
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3716..4088
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3745..4333)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4342..4375)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4543..5130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4622..4641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5143..5934,5935..6003))
                     /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     5766..5785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     6246..7073
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             6246..6370
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6408..7013)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
       61 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      121 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      181 acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc
      241 cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta
      301 cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacacca atgggcgtgg
      361 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      421 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac
      481 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagacgtc gtttagtgaa
      541 ccgtcagatc actagatgct ttattgcggt agtttatcac agttaaattg ctaacgccag
      601 tctcgaactt aacgtgcaga agttggtcgt gaggcactgg gcaggtaagt atcgggccct
      661 ttgtgcgggg ggagcggctc ggggctgtcc gcggggggac ggctgccttc gggggggacg
      721 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
      781 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc
      841 tcatcatttt ggcaaagaat tggatcggac cgaaattaat acgactcact ataggggaat
      901 tgtgagcgga taacaattcc ccggagttaa tccgggacct ttaattcaac ccaacacaat
      961 atattatagt taaataagaa ttattatcaa atcatttgta tattaattaa aatactatac
     1021 tgtaaattac attttattta caatcaaagg agatatacca tggcacacca tcaccaccac
     1081 cacgggagcg gggagcagaa actaatatca gaggaggatc ttggctcagg ttcaggatcc
     1141 gacggcgtgc tgaagggcga cgtcaagcac aagctgctgc tggagggcgg cggccaccac
     1201 cgcgttgact tcaagaccat ctacagggcc aagaaggcgg tgaagctgcc cgactatcac
     1261 tttgtggacc accgcatcga gatcctgaac cacgacaagg actacaacaa ggtgaccgtt
     1321 tacgagagcg ccgtggcccg caactccacc gacggcatgg acgagctgta caagggtgct
     1381 agcggaggaa tggtgagcaa gggcgaggag accacaatgg gcgtaatcaa gcccgacatg
     1441 aagatcaagc tgaagatgga gggcaacgtg aatggccacg ccttcgtgat cgagggcgag
     1501 ggcgagggca agccctacga cggcaccaac accatcaacc tggaggtgaa ggagggagcc
     1561 cccctgccct tctcctacga cattctgacc accgcgttcg cctacggcaa cagggccttc
     1621 accaagtacc ccgacgacat ccccaactac ttcaagcagt ccttccccga gggctactct
     1681 tgggagcgca ccatgacctt cgaggacaag ggcatcgtga aggtgaagtc cgacatctcc
     1741 atggaggagg actccttcat ctacgagata cacctcaagg gcgagaactt cccccccaac
     1801 ggccccgtga tgcagaagaa gaccaccggc tgggacgcct ccaccgagag gatgtacgtg
     1861 cgctccggaa tggcggacga ggagaagctg ccgcccggct gggagaagcg catgagccgc
     1921 agctcaggcc gagtgtacta cttcaaccac atcactaacg ccagccagtg ggagcggccc
     1981 agcggcaaca gcagcagtgg tggcaaaaac gggcaggggg agcctgccag gaagctgcgc
     2041 attctgcagt cgactaggga gcaagggctt gtgcccgggt ctagaggcgg aggtggtgga
     2101 ggaggtggag gtggtggagg tggaggtgga ggtggtggcg gtggtggagg aggaggagct
     2161 agaggcggag gtggtggagg aggtggaggt ggtggaggtg gaggtggagg tggtggcggt
     2221 ggtggaggag gaggagccag cggcggaggt ggtggaggag gtggaggtgg tggaggtgga
     2281 ggtggaggtg gtggcggtgg tggaggagga ggaaccggtg ctgaccaact gactgaagag
     2341 tggtcgacgg tacagcggac cgcgcctgac gtaccacgta caccagtagg aaaggcaaag
     2401 ctttcattcc aattcccgcg ggcgcgggac atcgcggccg cgatggacgg cggcgtgcag
     2461 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2521 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2581 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2641 aagggtggca gcggtggcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2701 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2761 gagggcgatg ccacctacgg caagctgacc ctgaagctga tctgcaccac cggcaagctg
     2821 cccgtgccct ggcccaccct cgtgaccacc ctgggctacg gcctgatgtg cttcgcccgc
     2881 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2941 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     3001 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3061 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc
     3121 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgagggt
     3181 accgatatcc ttcaaaaaaa acttgaagaa cttgaacttg acgaatgaga tatctaatga
     3241 ctcgagcacc accatcacca tcaccatcac taagtgatta acctcaggtg caggctgcct
     3301 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcgat
     3361 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     3421 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     3481 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     3541 agtttggcaa catatgccca tatgtaacta gcataacccc ttggggcctc taaacgggtc
     3601 ttgaggggtt ttttgctgaa agcatgcgga ggaaattctc cttgaagttt ccctggtgtt
     3661 caaagtaaag gagtttgcac cagacgcacc tctgttcact ggtccggcgt attaaaacac
     3721 gatacattgt tattagtaca tttattaagc gctagattct gtgcgttgtt gatttacaga
     3781 caattgttgt acgtatttta ataattcatt aaatttataa tctttagggt ggtatgttag
     3841 agcgaaaatc aaatgatttt cagcgtcttt atatctgaat ttaaatatta aatcctcaat
     3901 agatttgtaa aataggtttc gattagtttc aaacaagggt tgtttttccg aaccgatggc
     3961 tggactatct aatggatttt cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa
     4021 tctagctttg tcgatattcg tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa
     4081 aatattatgc gcttttgtat ttctttcatc actgtcgtta gtgtacaatt gactcgacgt
     4141 aaacacgtta aatagagctt ggacatattt aacatcgggc gtgttagctt tattaggccg
     4201 attatcgtcg tcgtcccaac cctcgtcgtt agaagttgct tccgaagacg attttgccat
     4261 agccacacga cgcctattaa ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga
     4321 gctttttgga attgcgatcg cataacttcg tatagcatac attatacgaa gttataagct
     4381 cggaacgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4561 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4621 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4681 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4741 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4801 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4861 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4921 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4981 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5041 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5101 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgttaccaat gcttaatcag
     5161 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5221 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5281 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5341 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5401 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5461 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5521 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5581 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5641 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5701 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5761 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5821 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5881 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5941 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6001 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgtccgc
     6061 gcgtttcctg catcttttaa tcaaatccca agatgtgtat aaacgcgccg gtatgtacag
     6121 gaagaggttt atactaaact gttacattgc aaacgtggtt tcgtgtgcca agtgtgaaaa
     6181 ccgatgttta atcaaggctc tgacgcattt ctacaaccac gactccaagt gtgtgggtga
     6241 agtcatgcat cttttaatca aatcccaaga tgtgtataaa ccaccaaact gccaaaaaat
     6301 gaaaactgtc gacaagctct gtccgtttgc tggcaactgc aagggtctca atcctatttg
     6361 taattattga ataataaaac aattataaat gtcaaatttg ttttttatta acgatacaaa
     6421 ccaaacgcaa caagaacatt tgtagtatta tctataattg aaaacgcgta gttataatcg
     6481 ctgaggtaat atttaaaatc attttcaaat gattcacagt taatttgcga caatataatt
     6541 ttattttcac ataaactaga cgccttgtcg tcttcttctt cgtattcctt ctctttttca
     6601 tttttctctt cataaaaatt aacatagtta ttatcgtatc catatatgta tctatcgtat
     6661 agagtaaatt ttttgttgtc ataaatatat atgtcttttt taatggggtg tatagtaccg
     6721 ctgcgcatag tttttctgta atttacaaca gtgctatttt ctggtagttc ttcggagtgt
     6781 gttgctttaa ttattaaatt tatataatca atgaatttgg gatcgtcggt tttgtacaat
     6841 atgttgccgg catagtacgc agcttcttct agttcaatta caccattttt tagcagcacc
     6901 ggattaacat aactttccaa aatgttgtac gaaccgttaa acaaaaacag ttcacctccc
     6961 ttttctatac tattgtctgc gagcagttgt ttgttgttaa aaataacagc cattgtaatg
     7021 agacgcacaa actaatatca caaactggaa atgtctatca atatatagtt gctgatggcc
     7081 ggcctattaa tagtaatcaa ttacggggtc attagttca
//
