LOCUS       sequence-362827-        7250 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cytosolic EBFP2 expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7250)
  AUTHORS   Yang JM, Chi WY, Liang J, Takayanagi S, Iglesias PA, Huang CH
  TITLE     Deciphering cell signaling networks with massively multiplexed 
            biosensor barcoding.
  JOURNAL   Cell. 2021 Dec 9;184(25):6193-6206.e14. doi: 
            10.1016/j.cell.2021.11.005. Epub 2021 Nov 26.
  PUBMED    34838160
REFERENCE   2  (bases 1 to 7250)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2021.11.005"; journalName: "Cell"; date: "2021-12-9-
            9"; volume: "184"; issue: "25"; pages: "6193-6206.e14"
COMMENT     Sequence Label: pVITRO1-BFP-NES
FEATURES             Location/Qualifiers
     source          1..7250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        51..242
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(74..94)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        256..1562
                     /label=mEF-1-alpha promoter
                     /note="strong constitutive promoter for mouse elongation 
                     factor EF-1-alpha"
     intron          583..1530
                     /label=mEF-1-alpha intron
                     /note="intron upstream of the start codon of mouse 
                     EF-1-alpha"
     polyA_signal    1607..1728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1618..1637)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1672..1691
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      1916..2504
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2405..2424
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2584..2887
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3002..4307
                     /label=rEF-1-alpha promoter
                     /note="strong constitutive promoter for rat elongation 
                     factor EF-1-alpha"
     misc_feature    3344..4275
                     /label=rEF-1-alpha intron
                     /note="intron upstream of the start codon of rat 
                     EF-1-alpha"
     regulatory      4314..4323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4320..4322,4323..4325,4326..5036)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(4365..4386)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    5097..5541
                     /label=FMDV IRES
                     /note="internal ribosome entry site (IRES) of the 
                     foot-and-mouth disease virus"
     promoter        5574..5620
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5612..5630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5640..6665
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    6677..7249
                     /label=EF-1-alpha poly(A) signal
                     /note="human EF-1-alpha polyadenylation signal"
ORIGIN
        1 cctgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
       61 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
      121 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
      181 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
      241 ccactagtgg agccgagagt aattcataca aaaggaggga tcgccttcgc aaggggagag
      301 cccagggacc gtccctaaat tctcacagac ccaaatccct gtagccgccc cacgacagcg
      361 cgaggagcat gcgctcaggg ctgagcgcgg ggagagcaga gcacacaagc tcatagaccc
      421 tggtcgtggg ggggaggacc ggggagctgg cgcggggcaa actgggaaag cggtgtcgtg
      481 tgctggctcc gccctcttcc cgagggtggg ggagaacggt atataagtgc ggcagtcgcc
      541 ttggacgttc tttttcgcaa cgggtttgcc gtcagaacgc aggtgagggg cgggtgtggc
      601 ttccgcgggc cgccgagctg gaggtcctgc tccgagcggg ccgggccccg ctgtcgtcgg
      661 cggggattag ctgcgagcat tcccgcttcg agttgcgggc ggcgcgggag gcagagtgcg
      721 aggcctagcg gcaaccccgt agcctcgcct cgtgtccggc ttgaggccta gcgtggtgtc
      781 cgcgccgccg ccgcgtgcta ctccggccgc actctggtct tttttttttt tgttgttgtt
      841 gccctgctgc cttcgattgc cgttcagcaa taggggctaa caaagggagg gtgcggggct
      901 tgctcgcccg gagcccggag aggtcatggt tggggaggaa tggagggaca ggagtggcgg
      961 ctggggcccg cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgg
     1021 ggtttttccc gaagcaacca ggctggggtt agcgtgccga ggccatgtgg ccccagcacc
     1081 cggcacgatc tggcttggcg gcgccgcgtt gccctgcctc cctaactagg gtgaggccat
     1141 cccgtccggc accagttgcg tgcgtggaaa gatggccgct cccgggccct gttgcaagga
     1201 gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg
     1261 aagagggcct ggtccctcac cggctgctgc ttcctgtgac cccgtggtcc tatcggccgc
     1321 aatagtcacc tcgggctttt gagcacggct agtcgcggcg gggggagggg atgtaatggc
     1381 gttggagttt gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt
     1441 catttttgga atttgtcccc ttgagttttg agcggagcta attctcgggc ttcttagcgg
     1501 ttcaaaggta tcttttaaac ccttttttag gtgttgtgaa aaccaccgct aattcaaagc
     1561 aaccggtgat atcaaagatc ttgtacagct agctggccag acatgataag atacattgat
     1621 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1681 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1741 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1801 aacctctaca aatgtggtat ggaaatgtta attaactagc catgaccaaa atcccttaac
     1861 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1921 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1981 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2041 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     2101 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2161 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2221 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2281 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2341 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2401 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2461 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2521 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttctta attaacctgc
     2581 aggcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     2641 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     2701 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     2761 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     2821 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     2881 taccatgatg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     2941 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttgac tagtggagcc
     3001 gagagtaatt catacaaaag gagggatcgc cttcgcaagg ggagagccca gggaccgtcc
     3061 ctaaattctc acagacccaa atccctgtag ccgccccacg acagcgcgag gagcatgcgc
     3121 ccagggctga gcgcgggtag atcagagcac acaagctcac agtccccggc ggtgggggga
     3181 ggggcgcgct gagcgggggc cagggagctg gcgcggggca aactgggaaa gtggtgtcgt
     3241 gtgctggctc cgccctcttc ccgagggtgg gggagaacgg tatataagtg cggtagtcgc
     3301 cttggacgtt ctttttcgca acgggtttgc cgtcagaacg caggtgagtg gcgggtgtgg
     3361 cttccgcggg ccccggagct ggagccctgc tctgagcggg ccgggctgat atgcgagtgt
     3421 cgtccgcagg gtttagctgt gagcattccc acttcgagtg gcgggcggtg cgggggtgag
     3481 agtgcgaggc ctagcggcaa ccccgtagcc tcgcctcgtg tccggcttga ggcctagcgt
     3541 ggtgtccgcc gccgcgtgcc actccggccg cactatgcgt tttttgtcct tgctgccctc
     3601 gattgccttc cagcagcatg ggctaacaaa gggagggtgt ggggctcact cttaaggagc
     3661 ccatgaagct tacgttggat aggaatggaa gggcaggagg ggcgactggg gcccgcccgc
     3721 cttcggagca catgtccgac gccacctgga tggggcgagg cctgtggctt tccgaagcaa
     3781 tcgggcgtga gtttagccta cctgggccat gtggccctag cactgggcac ggtctggcct
     3841 ggcggtgccg cgttcccttg cctcccaaca agggtgaggc cgtcccgccc ggcaccagtt
     3901 gcttgcgcgg aaagatggcc gctcccgggg ccctgttgca aggagctcaa aatggaggac
     3961 gcggcagccc ggtggagcgg gcgggtgagt cacccacaca aaggaagagg gccttgcccc
     4021 tcgccggccg ctgcttcctg tgaccccgtg gtctatcggc cgcatagtca cctcgggctt
     4081 ctcttgagca ccgctcgtcg cggcgggggg aggggatcta atggcgttgg agtttgttca
     4141 catttggtgg gtggagacta gtcaggccag cctggcgctg gaagtcattc ttggaatttg
     4201 cccctttgag tttggagcga ggctaattct caagcctctt agcggttcaa aggtattttc
     4261 taaacccgtt tccaggtgtt gtgaaagcca ccgctaattc aaagcaatcc ggagccacca
     4321 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4381 gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat gccaccaacg
     4441 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4501 tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc
     4561 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4621 tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4681 tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc ctggggcaca
     4741 agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag cagaagaacg
     4801 gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg cagctcgccg
     4861 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacagccact
     4921 acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4981 tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg tacaaaggaa
     5041 gctccctcca gaagaaactg gaggaactgg aactgtgagg atcccgtacg cctaggagca
     5101 ggtttcccca atgacacaaa acgtgcaact tgaaactccg cctggtcttt ccaggtctag
     5161 aggggtaaca ctttgtactg cgtttggctc cacgctcgat ccactggcga gtgttagtaa
     5221 cagcactgtt gcttcgtagc ggagcatgac ggccgtggga actcctcctt ggtaacaagg
     5281 acccacgggg ccaaaagcca cgcccacacg ggcccgtcat gtgtgcaacc ccagcacggc
     5341 gactttactg cgaaacccac tttaaagtga cattgaaact ggtacccaca cactggtgac
     5401 aggctaagga tgcccttcag gtaccccgag gtaacacgcg acactcggga tctgagaagg
     5461 ggactggggc ttctataaaa gcgctcggtt taaaaagctt ctatgcctga ataggtgacc
     5521 ggaggtcggc acctttcctt tgcaattact gaccctatga atacactgac tgtttgacaa
     5581 ttaatcatcg gcatagtata tcggcatagt ataatacgac tcactatagg agggccacca
     5641 tgaagaaacc tgaactgaca gcaacttctg ttgagaagtt tctcattgaa aaatttgatt
     5701 ctgtttctga tctcatgcag ctgtctgaag gtgaagaaag cagagccttt tcttttgatg
     5761 ttggaggaag aggttatgtt ctgagggtca attcttgtgc tgatggtttt tacaaagaca
     5821 gatatgttta cagacacttt gcctctgctg ctctgccaat tccagaagtt ctggacattg
     5881 gagaattttc tgaatctctc acctactgca tcagcagaag agcacaagga gtcactctcc
     5941 aggatctccc tgaaactgag ctgccagctg ttctgcaacc tgttgctgaa gcaatggatg
     6001 ccattgcagc agctgatctg agccaaacct ctggatttgg tccttttggt ccccaaggca
     6061 ttggtcagta caccacttgg agggatttca tttgtgccat tgctgatcct catgtctatc
     6121 actggcagac tgtgatggat gacacagttt ctgcttctgt tgctcaggca ctggatgaac
     6181 tcatgctgtg ggcagaagat tgtcctgaag tcagacacct ggtccatgct gattttggaa
     6241 gcaacaatgt tctgacagac aatggcagaa tcactgcagt cattgactgg tctgaagcca
     6301 tgtttggaga ttctcaatat gaggttgcca acattttttt ttggagacct tggctggctt
     6361 gcatggaaca acaaacaaga tattttgaaa gaagacaccc agaactggct ggttccccca
     6421 gactgagagc ctacatgctc agaattggcc tggaccaact gtatcaatct ctggttgatg
     6481 gaaactttga tgatgctgct tgggcacaag gaagatgtga tgccattgtg aggtctggtg
     6541 ctggaactgt tggaagaact caaattgcaa gaaggtctgc tgctgtttgg actgatggat
     6601 gtgttgaagt tctggctgac tctggaaaca ggagaccctc cacaagaccc agagccaagg
     6661 aatgaatatt agctagatta tccctaatac ctgccacccc actcttaatc agtggtggaa
     6721 gaacggtctc agaactgttt gtttcaattg gccatttaag tttagtagta aaagactggt
     6781 taatgataac aatgcatcgt aaaaccttca gaaggaaagg agaatgtttt gtggaccact
     6841 ttggttttct tttttgcgtg tggcagtttt aagttattag tttttaaaat cagtactttt
     6901 taatggaaac aacttgacca aaaatttgtc acagaatttt gagacccatt aaaaaagtta
     6961 aatgagaaac ctgtgtgttc ctttggtcaa caccgagaca tttaggtgaa agacatctaa
     7021 ttctggtttt acgaatctgg aaacttcttg aaaatgtaat tcttgagtta acacttctgg
     7081 gtggagaata gggttgtttt ccccccacat aattggaagg ggaaggaata tcatttaaag
     7141 ctatgggagg gttgctttga ttacaacact ggagagaaat gcagcatgtt gctgattgcc
     7201 tgtcactaaa acaggccaaa aactgagtcc ttgggttgca tagaaagctg
//
