LOCUS       sequence-177125-        5394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-SpvB (Salmonella SpvB 375-591) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5394)
  AUTHORS   Harterink M, da Silva ME, Will L, Turan J, Ibrahim A, Lang AE, van 
            Battum EY, Pasterkamp RJ, Kapitein LC, Kudryashov D, Barres BA, 
            Hoogenraad CC, Zuchero JB
  TITLE     DeActs: genetically encoded tools for perturbing the actin 
            cytoskeleton in single cells.
  JOURNAL   Nat Methods. 2017 Apr 10. doi: 10.1038/nmeth.4257.
  PUBMED    28394337
REFERENCE   2  (bases 1 to 5394)
  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: "Nat 
            Methods. 2017 Apr 10. doi: 10.1038/nmeth.4257."
COMMENT     Sequence Label: pCMV-DeAct-SpvB
FEATURES             Location/Qualifiers
     source          1..5394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2182..2303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2219..2238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2273..2292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2310..2765)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2447..2468)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2659..2678
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2792..2896
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2898..3255
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2898..3089
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2921..2941)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3106..3241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3290..4084
                     /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             3290..4084
                     /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(3344..3363)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3954..3973
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4272..4291)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4316..4363
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4692..5280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5181..5200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctatggg aggtaattca
     1381 tctcgaccaa aatcaaaatg ggcgattgta gaggcatcaa agcagattca agctctgagg
     1441 tactattcag ctcaagggta cagtgtgatt aataaatatt tacgtgggga tgattatcct
     1501 gaaacacagg caaaagaaac tctgctctcc agagactatc tttccacaaa tgaacccagt
     1561 gatgaggagt ttaaaaatgc catgtcagtt tatataaatg atattgcgga gggattaagt
     1621 tcacttcccg aaacagatca cagagtcgta taccggggcc tgaagcttga taagcccgca
     1681 ttatcggatg tgctgaagga atacactact ataggtaata taataataga taaagctttt
     1741 atgagtacat cgccagataa ggcatggata aatgacacta ttctcaacat atacctagaa
     1801 aaaggacata aaggtagaat actcggagat gttgcacatt ttaagggaga ggcagagatg
     1861 cttttccctc caaatactaa actcaaaatc gaaagcattg taaattgtgg atcccaagac
     1921 tttgcaagcc agcttagtaa gctgagatta agtgatgatg caactgctga cacaaacagg
     1981 ataaaaagaa taataaacat gagggtactc aactcatgat taaattctgc agtcgacggt
     2041 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt
     2101 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2161 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2221 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2281 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt
     2341 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2401 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2461 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2521 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2581 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2641 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2701 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2761 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2821 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2881 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     2941 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3001 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3061 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3121 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3181 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3241 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3301 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3361 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3421 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     3481 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3541 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3601 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3661 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3721 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3781 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     3841 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3901 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3961 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4021 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4081 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4141 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4201 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     4261 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     4321 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     4381 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     4441 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     4501 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     4561 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4621 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4681 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4741 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4801 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4861 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4921 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4981 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5041 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5101 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5161 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5221 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5281 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5341 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
