LOCUS       sequence-484512-        6331 bp    DNA     circular SYN 27-AUG-2025
DEFINITION  Encodes human WASP and N-terminal EGFP tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6331)
  AUTHORS   Qi Y, Yu CH
  TITLE     PI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome 
            assembly.
  JOURNAL   Cell Mol Life Sci. 2025 Mar 24;82(1):127. doi: 
            10.1007/s00018-025-05664-2.
  PUBMED    40126693
REFERENCE   2  (bases 1 to 6331)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 27, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Mol 
            Life Sci."; date: "2025-03-24"; volume: "82(1):127. doi"; pages: " 
            10.1007/s00018-025-05664-2"
COMMENT     Sequence Label: EGFP-WASP-3D
FEATURES             Location/Qualifiers
     source          1..6331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1316..1318,1319..1321,1322..2032)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1316..1318,1319..1321,1322..2032)
                     /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(1361..1382)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1622..1641)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1969..1990
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3822..3943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3859..3878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3913..3932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3950..4405)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4087..4108)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4299..4318
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4432..4536
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4538..4895
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4538..4729
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4561..4581)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4746..4881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4930..5724
                     /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             4930..5724
                     /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(4984..5003)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5594..5613
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5912..5931)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5956..6003
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     1321 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1381 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1441 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1501 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1561 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1621 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1681 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1741 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1801 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1861 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1921 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1981 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggccg
     2041 ctctagagaa agcaccatga gtgggggccc aatgggagga aggcccgggg gccgaggagc
     2101 accagcggtt cagcagaaca taccctccac cctcctccag gaccacgaga accagcgact
     2161 ctttgagatg cttggacgaa aatgcttgac gctggccact gcagttgttc agctgtacct
     2221 ggcgctgccc cctggagctg agcactggac caaggagcat tgtggggctg tgtgcttcgt
     2281 gaaggataac ccccagaagt cctacttcat ccgcctttac ggccttcagg ctggtcggct
     2341 gctctgggaa caggagctgt actcacagct tgtctactcc acccccaccc ccttcttcca
     2401 caccttcgct ggagatgact gccaagcggg gctgaacttt gcagacgagg acgaggccca
     2461 ggccttccgg gccctcgtgc aggagaagat acaaaaaagg aatcagaggc aaagtggaga
     2521 cagacgccag ctacccccac caccaacacc agccaatgaa gagagaagag gagggctccc
     2581 acccctgccc ctgcatccag gtggagacca aggaggccct ccagtgggtc cgctctccct
     2641 ggggctggcg acagtggaca tccagaaccc tgacatcacg agttcacgat accgtgggct
     2701 cccagcacct ggacctagcc cagctgataa gaaacgctca gggaagaaga agatcagcaa
     2761 agctgatatt ggtgcaccca gtggagacaa ggatgtcagc gacgtggggt gggaccccca
     2821 gaatggattt gacgtgaaca acctcgaccc agatctgcgg agtctgttct ccagggcagg
     2881 aatcagcgag gcccagctca ccgacgccga gacctctaaa cttatctacg acttcattga
     2941 ggaccagggt gggctggagg ctgtgcggca ggagatgagg cgccaggagc cacttccgcc
     3001 gcccccaccg ccatctcgag gagggaacca gctcccccgg ccccctattg tggggggtaa
     3061 caagggtcgt tctggtccac tgccccctgt acctttgggg attgccccac ccccaccaac
     3121 accccgggga cccccacccc caggccgagg gggccctcca ccaccacccc ctccagctac
     3181 tggacgttct ggaccactgc cccctccacc ccctggagct ggtgggccac ccatgccacc
     3241 accaccgcca ccaccgccac cgccgcccag ctccgggaat ggaccagccc ctcccccact
     3301 ccctcctgct ctggtgcctg ccgggggcct ggcccctggt gggggtcggg gagcgctttt
     3361 ggatcaaatc cggcagggaa ttcagctgaa caagacccct ggggccccag agagctcagc
     3421 gctgcagcca ccacctcaga gctcagaggg actggtgggg gccctgatgc acgtgatgca
     3481 gaagagaagc agagccatcc actcctccga cgaaggggag gaccaggctg gcgatgaaga
     3541 tgaagatgat gaatgggatg actgagtggc tgagttactt gctgccctgt gctcctcccc
     3601 gcaggacatg gctccccctc cacctgctct gtgccgggct gcaggaattc gatatcaagc
     3661 ttatcgatac cgtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca
     3721 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3781 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3841 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3901 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     3961 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4021 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     4081 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4141 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4201 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4261 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4321 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4381 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4441 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4501 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     4561 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4621 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4681 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4741 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4801 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4861 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4921 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4981 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     5041 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     5101 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     5161 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     5221 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5281 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5341 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5401 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     5461 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5521 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5581 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5641 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5701 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5761 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5821 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5881 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5941 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     6001 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     6061 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     6121 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     6181 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6241 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6301 cagaccccgt agaaaagatc aaaggatctt c
//
