LOCUS       sequence-310129-        5008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Alternate MPAct using Lyn/LifeAct instead of CaaX/F-tractin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5008)
  AUTHORS   Bisaria A, Hayer A, Garbett D, Cohen D, Meyer T
  TITLE     Membrane-proximal F-actin restricts local membrane protrusions and 
            directs cell migration.
  JOURNAL   Science. 2020 Jun 12;368(6496):1205-1210. doi: 
            10.1126/science.aay7794.
  PUBMED    32527825
REFERENCE   2  (bases 1 to 5008)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2020-06-12- 12"; volume: "368"; 
            issue: "6496"; pages: "1205-1210"
COMMENT     Sequence Label: C1-Lyn-mCitrine-EEVEE-LifeAct
FEATURES             Location/Qualifiers
     source          1..5008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(285..304)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    329..376
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      705..1293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1194..1213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1468..1771
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1772..1975
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1926..1946
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(2107..2109,2110..2820)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2149..2170)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2410..2429)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2757..2778
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3028..3078
                     /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"
     polyA_signal    3203..3324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3240..3259)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3294..3313
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3331..3786)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3468..3489)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3680..3699
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3813..3917
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3919..4276
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3919..4110
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3942..3962)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4127..4262
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4311..5008,1..97)
                     /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             join(4311..5008,1..97)
                     /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(4365..4384)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4975..4994
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
       61 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
      121 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
      181 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
      241 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
      301 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
      361 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
      421 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
      481 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
      541 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
      601 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
      661 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      721 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      781 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      841 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      901 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      961 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1021 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1081 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1141 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1201 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1261 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1321 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1381 gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt
     1441 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1501 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1561 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1621 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1681 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1741 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1801 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1861 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1921 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
     1981 agtgaaccgt cagatccgct agcgctaccg gactcagatc tcgagctcaa gcttatggga
     2041 tgtataaaat caaaagggaa agacagcgcg ggagcagaca gtgctggctc cgctggtagt
     2101 gctggtgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2161 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2221 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2281 accctcgtga ccaccttcgg ctacggcctg atgtgcttcg cccgctaccc cgaccacatg
     2341 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2401 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2461 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2521 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2581 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2641 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2701 cactacctga gctaccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     2761 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaaa
     2821 agaagccgag ctcaagcttc gaccggtggt ggatccggag gaaccagtgc tggcgggagc
     2881 gctggtggaa gtgcgggggg atcttctgct ggaggcagtg ctggagggtc aggctctgct
     2941 ggcgggtctg ctggagggag tacatcagcg ggaggttccg ctgggggcag cgccggagga
     3001 tctgcaggcg ggagtgccgg aggcactatg ggcgtggcgg acctgatcaa aaaatttgag
     3061 agcatctcaa aagaggaata agcggccgcg actctagatc ataatcagcc ataccacatt
     3121 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     3181 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     3241 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3301 gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat
     3361 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3421 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     3481 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     3541 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     3601 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     3661 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3721 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3781 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3841 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3901 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4081 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4141 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4201 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4261 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4321 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4381 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4441 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     4501 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4561 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4621 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4681 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4741 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4801 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     4861 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4921 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4981 ctacccgtga tattgctgaa gagcttgg
//
