LOCUS       sequence-388854-        7709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RAPH1 fragment (1-535) tagged to eGFP in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7709)
  AUTHORS   Popovic A, Miihkinen M, Ghimire S, Saup R, Gronloh MLB, Ball NJ, 
            Goult BT, Ivaska J, Jacquemet G
  TITLE     Myosin-X recruits lamellipodin to filopodia tips.
  JOURNAL   J Cell Sci. 2023 Mar 1;136(5):jcs260574. doi: 10.1242/jcs.260574. 
            Epub 2023 Mar 2.
  PUBMED    36861887
REFERENCE   2  (bases 1 to 7709)
  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: "J Cell 
            Sci."; date: "2023-03-1"; volume: "136(5):jcs260574. doi"; pages: " 
            10.1242/jcs.260574. Epub 2023 Mar 2"
COMMENT     Sequence Label: eGFP-RAPH1-F1
FEATURES             Location/Qualifiers
     source          1..7709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             923..1636
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(923..925,926..1636)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(965..986)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1226..1245)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1573..1594
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3300..3317)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3306..3530
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3576..4004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3873..3894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4013..4033)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4018..4347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4198..4333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4260..4279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4414..5208
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4468..4487)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5078..5097
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5382..5503
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5419..5438)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5473..5492
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5552..5568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5552..5568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5565..5587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5576..5592
                     /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)."
     promoter        complement(join(5600..5606,5607..5624,5625..5630))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5645..5666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5783..5800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5954..6542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6034..6053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6713..7504,7505..7573))
                     /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     7336..7355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7574..7678)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      961 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1021 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1081 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1141 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1201 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1261 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1321 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1381 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1441 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1501 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1561 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat
     1621 ggacgagctg tacaagggta ccgagctcgg atccatggag cagctatcag atgaagaaat
     1681 tgatcatggt gctgaagaag acagtgacaa ggaagatcag gacctggaca aaatgtttgg
     1741 agcctggctt ggagaactag acaaactcac tcagagtttg gattctgaca agcccatgga
     1801 accagtaaaa agatctcctc ttcgccagga aacaaacatg gccaactttt cttaccgctt
     1861 ctccatatac aacttgaatg aagctctgaa tcagggagag actgtggatc tggatgcctt
     1921 gatggctgat ctttgctcta tagagcagga gctcagcagc attggttcag gaaacagtaa
     1981 gcgtcaaatc acagaaacga aagctactca gaaattgcct gttagccgac atacattgaa
     2041 acatggcacc ttgaaaggat tatcttcttc atctaatagg atagctaaac cttcccatgc
     2101 cagctactcc ttggacgacg tcactgcaca gttagaacag gcctctttga gtatggatga
     2161 ggctgctcag caatctgtac tagaagatac taaaccctta gtaactaatc agcacagaag
     2221 aaccgcgtca gcaggcacag tgagtgatgc tgaagtacac tctattagta attcctccca
     2281 ttccagcatc acttccgcag cctccagcat ggactctttg gatattgata aagtaacacg
     2341 ccctcaagag ctggatttga cacatcaagg gcagccaatt actgaggaag aacaggcagc
     2401 aaaattgaaa gctgagaaga tcagagttgc cctagagaaa attaaagagg cacaagtgaa
     2461 aaagctggtg atcagagtcc acatgtctga tgacagttct aaaacaatga tggtggatga
     2521 gaggcagaca gtaagacaag tactggataa cctgatggac aaatcccact gcggttatag
     2581 tttagactgg tcactggtag aaaccgtttc tgaattacaa atggagagaa tctttgaaga
     2641 ccatgaaaac ttggttgaaa atcttcttaa ttggacaaga gatagccaaa acaagcttat
     2701 atttatggag cgtatagaaa aatatgcact tttcaaaaac ccacagaatt atcttttggg
     2761 gaaaaaagaa acagctgaga tggcagatag aaacaaagaa gtcctcttgg aggaatgttt
     2821 ttgtggaagt tctgtaactg taccagaaat tgaaggagtc ctttggttga aggatgatgg
     2881 caagaagtcc tggaaaaagc gttattttct cttgcgagca tctggtatct actatgttcc
     2941 caaaggaaaa gcaaaggtct ctcgggatct ggtgtgcttt ctccagctgg atcatgtcaa
     3001 cgtttattat ggccaggact atcggaacaa atacaaagca cctacagact attgtctggt
     3061 gctgaagcat ccacaaatcc agaagaaatc tcaatatatc aaataccttt gttgtgatga
     3121 tgtgaggaca ctgcatcagt gggtcaatgg gttccgcatt gcaaagtatg ggaagcagct
     3181 ctatatgaac taccaagaag ccttgaagag gacagagtca gcctatgatt ggacttcctt
     3241 atccagctcc agcattaaat agctcgagtc tagagggccc gtttaaaccc gctgatcagc
     3301 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3361 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3421 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3481 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3541 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3601 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3661 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3721 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3781 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3841 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3901 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3961 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     4021 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4081 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4141 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4201 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4261 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4321 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4381 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4441 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4501 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4561 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4621 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4681 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4741 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4801 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4861 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4921 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4981 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5041 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5101 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5161 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5221 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5281 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5341 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5401 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5461 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5521 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5581 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5641 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5701 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5761 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5821 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5881 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5941 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6001 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6061 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6121 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6181 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6241 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6301 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6361 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6421 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6481 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6541 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6601 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6661 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6721 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6781 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6841 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6901 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6961 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7021 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7081 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7141 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7201 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7261 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7321 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7381 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7441 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7501 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7561 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7621 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7681 catttccccg aaaagtgcca cctgacgtc
//
