LOCUS       sequence-340326-        7397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoding EKAREV-NLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  AUTHORS   Lin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, Aoki K, 
            Iwamoto R, Terai K, Matsuda M
  TITLE     Redundant roles of EGFR ligands in the ERK activation waves during 
            collective cell migration.
  JOURNAL   Life Sci Alliance. 2021 Oct 19;5(1). pii: 5/1/e202101206. doi: 
            10.26508/lsa.202101206. Print 2022 Jan.
  PUBMED    34667080
REFERENCE   2  (bases 1 to 7397)
  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: "Life Sci 
            Alliance."; date: "2021-10-19"; volume: "5(1). pii: 5/1/e202101206. 
            doi"; pages: " 10.26508/lsa.202101206. Print 2022 Jan"
COMMENT     Sequence Label: pT2A-EKAREV-NLS
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(125..193,194..985)
                     /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     complement(343..362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1156..1744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1645..1664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1898..1915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2032..2053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2068..2073,2074..2091,2092..2098)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2106..2122
                     /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)."
     primer_bind     2111..2133
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2130..2146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2130..2146
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2165..2185
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2167..2185
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(2242..2253,2254..2441)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     enhancer        2463..2842
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          3122..4138
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4062..4084
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4146..4165
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(4196..4198,4199..4201,4202..4909)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     CDS             5084..5431
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             join(5501..5503,5504..5506,5507..6019)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             6218..6238
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6290..6424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6314..6333)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6368..6387
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(6546..6708,6709..6720)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6750..6769)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6751..6769)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6779..6796)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6779..6795)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6788..6810)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6937..7364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7023..7042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7233..7254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
       61 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      121 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      181 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      241 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      301 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      361 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      421 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      481 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      541 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      601 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      661 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      721 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      781 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      841 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      901 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
      961 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1021 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1081 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1801 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1861 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1921 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1981 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2041 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2101 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctcggatctg ctgggcttgc
     2221 tgaaggtagg gggtcaagaa ccagaggtgt aaagtacttg agtaatttta cttgattact
     2281 gtacttaagt attatttttg gggattttta ctttacttga gtacaattaa aaatcaatac
     2341 ttttactttt acttaattac atttttttag aaaaaaaagt actttttact ccttacaatt
     2401 ttatttacag tcaaaaagta cttatttttt ggagatcact tgggccctct agatgcatgc
     2461 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2521 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2581 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2641 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2701 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2761 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2821 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     2881 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     2941 gatgggggcg gggggggggg gggggcgcgc gccrggsggg gsggggsggg gsggggggsg
     3001 gggsggggsg rggcggrgag gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     3061 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     3121 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     3181 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     3241 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     3301 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
     3361 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
     3421 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     3481 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     3541 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     3601 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     3661 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     3721 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     3781 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     3841 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     3901 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     3961 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     4021 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     4081 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     4141 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcatggt
     4201 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     4261 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     4321 gctgaccctg aagcttctat gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     4381 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     4441 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     4501 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     4561 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     4621 ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
     4681 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
     4741 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     4801 gagctaccag tccgccctgt tcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     4861 ggagttcctg accgccgccg ggatcactga gggcatgaac gagctgtacc tcgagatggc
     4921 ggacgaggag aagctgccgc ccggctggga gaagcgcatg agccgcagct caggccgagt
     4981 gtactacttc aaccacatca ctaacgccag ccagtgggag cggcccagcg gcaacagcag
     5041 cagtggtggc aaaaacgggc agggggagcc tgccaggggt accagtgctg gtggtagtgc
     5101 tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggttccggca gtgctggtgg
     5161 tagtgctggt ggtagtacca gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc
     5221 tggtggtagt gctggtggtt ccggcagtgc tggtggtagt gctggtggta gtaccagtgc
     5281 tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggttccgg
     5341 cagtgctggt ggtagtgctg gtggtagtac cagtgctggt ggtagtgctg gtggtagtgc
     5401 tggtggtagt gctggtggta gtgctggtgg ttccggacca gatgtcccta gaactccagt
     5461 ggataaagca aagctgtcat tccaatttcc gggcggccgc atggtgagca agggcgagga
     5521 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacag
     5581 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     5641 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgacctg
     5701 gggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5761 cgccatgccc gaaggctacg tccaggagcg taccatcttc ttcaaggacg acggcaacta
     5821 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     5881 gggcatcggc ttcaaggagg acggcaacat cctggggcac aagctagagt acaactacat
     5941 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg cccacttcaa
     6001 gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac
     6061 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     6121 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     6181 cgccgggatc actctcggca tggacgagct gtctagacct aagaagaagc gtaaggtgta
     6241 agtcgagcgg ccgccaccgc ggtggagctc gaattaattc atcgatgatg atccagacat
     6301 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     6361 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     6421 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     6481 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctagatca
     6541 gatctttctt gcttttactt ttacttcctt aatactcaag tacaatttta atggagtact
     6601 tttttacttt tactcaagta agattctagc cagatacttt tacttttaat tgagtaaaat
     6661 tttccctaag tacttgtact ttcacttgag taaaattttt gagtactttt tacacctctg
     6721 tcaagaacca tatgccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac
     6781 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     6841 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     6901 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     6961 gcgcggcggg tgtggtggtt acgcgcagcg tgaccaccac acccgccgcg ccctagcgcc
     7021 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     7081 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     7141 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     7201 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     7261 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     7321 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     7381 gcttacaatt taggtgg
//
