LOCUS       sequence-251686-        4882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains insert allowing in-vivo visualization of F-actin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4882)
  AUTHORS   Tran M, Dave M, Lansford R
  TITLE     Fast and Efficient Expression of Multiple Proteins in Avian Embryos 
            Using mRNA Electroporation.
  JOURNAL   J Vis Exp. 2019 Jun 7;(148). doi: 10.3791/59664.
  PUBMED    31233018
REFERENCE   2  (bases 1 to 4882)
  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: "J Vis 
            Exp."; date: "2019-06-7"; volume: "(148). doi"; pages: " 
            10.3791/59664"
COMMENT     Sequence Label: pCS2+Lifeact-eGFP
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1083..1133
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      1149..1158
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1155..1157,1158..1160,1161..1874)
                     /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
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1200..1221)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1461..1480)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1808..1829
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1912..2046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1936..1955)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1990..2009
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2159..2179)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2159..2177)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2198..2214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2198..2214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2211..2233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2222..2238
                     /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(2246..2252,2253..2270,2271..2276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2291..2312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2429..2446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2600..3188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2680..2699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3359..4150,4151..4219))
                     /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     3982..4001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4220..4324)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4350..4805)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4487..4508)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4699..4718
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ctcgagctca agcttcgaat
     1081 tcatgggtgt cgcagatttg atcaagaaat tcgaaagcat ctcaaaggaa gaaggggatc
     1141 caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1201 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1261 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1321 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1381 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1441 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1501 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1561 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1621 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1681 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1741 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     1801 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1861 agctgtacaa gtaagtcgag cctctagaac tatagtgagt cgtattacgt agatccagac
     1921 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     1981 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2041 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     2101 gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc ttttgttccc
     2161 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2221 attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct
     2281 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2341 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2401 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2461 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2521 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2581 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2641 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2701 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2761 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2821 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2881 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2941 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3001 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3061 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3121 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3181 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3241 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3301 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3361 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3421 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3481 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3541 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3601 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3661 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3721 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3781 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3841 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3901 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3961 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4021 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4081 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4141 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4201 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4261 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4321 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     4381 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4441 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4501 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4561 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     4621 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4681 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4741 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4801 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4861 ttcgctatta cgccagtcga cc
//
