LOCUS       sequence-482896-        4692 bp    DNA     circular SYN 12-AUG-2025
DEFINITION  Localization: Actin, Excitation: 505 / 569, Emission: 516 / 581.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4692)
  AUTHORS   Paez-Segala MG, Sun MG, Shtengel G, Viswanathan S, Baird MA, Macklin
            JJ, Patel R, Allen JR, Howe ES, Piszczek G, Hess HF, Davidson MW, 
            Wang Y, Looger LL
  TITLE     Fixation-resistant photoactivatable fluorescent proteins for CLEM.
  JOURNAL   Nat Methods. 2015 Jan 12. doi: 10.1038/nmeth.3225.
  PUBMED    25581799
REFERENCE   2  (bases 1 to 4692)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 12, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2015 Jan 12. doi: 10.1038/nmeth.3225."
COMMENT     Sequence Label: mEos4b-Lifeact-7
FEATURES             Location/Qualifiers
     source          1..4692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      537..546
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             543..593
                     /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      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             615..1298
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /translation="MVSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEV
                     KEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNA
                     RNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALL
                     LEGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLP
                     DNARR"
     polyA_signal    1420..1541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1457..1476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1511..1530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1548..2003)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1685..1706)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1897..1916
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2030..2134
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2136..2493
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2136..2327
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2159..2179)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2344..2479
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2528..3322
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2528..3322
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2582..2601)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3192..3211
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3510..3529)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3554..3601
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3930..4518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4419..4438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      541 ccatgggcgt ggccgacttg atcaagaagt tcgagtccat ctccaaggag gagggggatc
      601 caccggtcgc caccatggtg agtgcgatta agccagacat gaggatcaaa ctccgtatgg
      661 aaggcaacgt aaacgggcac cactttgtga tcgacggaga tggtacaggc aagccttatg
      721 agggaaaaca gaccatggat cttgaagtca aagagggcgg acctctgcct tttgcctttg
      781 atatcctgac cactgcattc cattacggca acagggtatt cgtgaaatat ccagacaaca
      841 tacaagacta ttttaagcag tcgtttccta aggggtattc gtgggaacga agcttgactt
      901 tcgaagacgg gggcatttgc aatgccagaa acgacataac aatggaaggg gacactttct
      961 ataataaagt tcgattttat ggtaccaact ttcccgccaa tggtccagtt atgcagaaga
     1021 agacgctgaa atgggagccc tccactgaga aaatgtatgt gcgtgatgga gtgctgacgg
     1081 gtgatattga gatggctttg ttgcttgaag gaaatgccca ttaccgatgt gacttcagaa
     1141 ctacttacaa agctaaggag aagggtgtca agttaccagg cgcccacttt gtggaccacg
     1201 ccattgagat tttaagccat gacaaagatt acaacaaggt taagctgtat gagcatgctg
     1261 ttgctcattc tggattgcct gacaatgcca gacgataagc ggccgcgact ctagatcata
     1321 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     1381 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     1441 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1501 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt
     1561 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1621 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     1681 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1741 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     1801 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1861 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1921 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1981 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2041 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2101 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     2161 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2221 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2281 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2341 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2401 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     2461 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     2521 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2581 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2641 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     2701 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     2761 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     2821 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     2881 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     2941 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3001 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     3061 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3121 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3181 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3241 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3301 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3361 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3421 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3481 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     3541 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     3601 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     3661 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     3721 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     3781 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3841 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3901 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3961 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4021 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4081 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4141 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4201 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4261 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4321 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4381 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4441 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4501 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4561 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4621 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     4681 tatggagttc cg
//
