LOCUS       sequence-194450-        5784 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses PIF3 (1-100 aa) fused with mEGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5784)
  AUTHORS   Uda Y, Goto Y, Oda S, Kohchi T, Matsuda M, Aoki K
  TITLE     Efficient synthesis of phycocyanobilin in mammalian cells for 
            optogenetic control of cell signaling.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Oct 24. pii: 201707190. doi: 
            10.1073/pnas.1707190114.
  PUBMED    29078307
REFERENCE   2  (bases 1 to 5784)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2017 Oct 24. pii: 201707190. doi: 
            10.1073/pnas.1707190114."
COMMENT     Sequence Label: pCAGGS-PIF3-mEGFP
FEATURES             Location/Qualifiers
     source          1..5784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1606..1628
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2077..2079,2080..2082,2083..2796)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELSR"
     primer_bind     complement(2122..2143)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2383..2402)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2730..2751
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2806..2825)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2871..2926
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2925..2944)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3287..3303)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3287..3303)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3300..3322)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3311..3327
                     /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(3335..3341,3342..3359,3360..3365))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3380..3401
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3460..3655
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3506..3641
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3661..3795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3698..3717)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3752..3771
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3863..3880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4034..4622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4114..4133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4793..5584,5585..5653))
                     /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     5416..5435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5654..5758)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgatcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
       61 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      361 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      721 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      781 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      841 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
      901 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
      961 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     1021 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     1081 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1141 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1201 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1261 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1321 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1381 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1441 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1501 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1561 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1621 gtgtgaccgg cggctctagc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1741 accatgcctc tgtttgagct tttcaggctc accaaagcta agcttgaatc tgctcaagac
     1801 aggaaccctt ctccacctgt agatgaagtt gtggagctgg tgtgggaaaa tggtcagata
     1861 tcaactcaaa gtcagtcaag tagatcgagg aacattcctc caccacaagc aaactcttct
     1921 agagctagag agattggaaa tggctcaaag acgactatgg tggacgagat ccctatgtca
     1981 gtgccatcac taatgacggg tttgagtcaa gacgatgact ttgttccatg gttgaatcat
     2041 catctcgagg gaggatccgg aggttccggc ggccgcatgg tgagcaaggg cgaggagctg
     2101 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2161 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2221 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2281 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2341 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2401 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2461 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2521 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2581 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2641 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctt
     2701 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2761 gggatcactc tcggcatgga cgagctgtct agataagtcg acggatcttt ttccctctgc
     2821 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     2881 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     2941 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     3001 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     3061 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     3121 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     3181 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     3241 cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     3301 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3361 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3421 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac
     3481 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3541 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3601 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3661 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3721 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3781 tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3841 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3901 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3961 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4021 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4081 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4141 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4201 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4261 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4321 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4381 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4441 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4501 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4561 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4621 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4681 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4741 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4801 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4861 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4921 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4981 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5041 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5101 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5161 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5221 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5281 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5341 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5401 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5461 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5521 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5581 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5641 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5701 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5761 catttccccg aaaagtgcca cctg
//
