LOCUS       sequence-388857-        6668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RAPH1 fragment (1062-1250) tagged to eGFP in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6668)
  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 6668)
  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-F4
FEATURES             Location/Qualifiers
     source          1..6668
                     /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"
     regulatory      2051..2060
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2259..2276)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2265..2489
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2535..2963
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2622..2641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2832..2853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2972..2992)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2977..3306
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3157..3292
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3219..3238
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3373..4167
                     /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(3427..3446)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4037..4056
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4341..4462
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4378..4397)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4432..4451
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4511..4527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4511..4527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4524..4546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4535..4551
                     /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(4559..4565,4566..4583,4584..4589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4604..4625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4742..4759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4913..5501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4993..5012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5672..6463,6464..6532))
                     /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     6295..6314
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6533..6637)
                     /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 atccgaattt ccttctcctc catccgattc
     1681 tgattttcca ccccctccac ctgaaacaga gcttcctctg ccccccattg agattccagc
     1741 agttttctcg ggaaacacct ctccaaaagt ggcagtcgtt aatcctcaac cacaacaatg
     1801 gtctaaaatg tcagtgaaga aggcccctcc acccacacga cccaaacgga atgatagcac
     1861 ccgcctcact caagctgaga tttctgagca gccaacaatg gccacagttg tgccacaagt
     1921 gcccacctct cccaaatcca gccttagtgt ccagcctgga ttcctggctg acctcaacag
     1981 gacactgcaa cgaaagtcca tcactcggca cggctcactc tcctcccgca tgtccagagc
     2041 agaaccaaca gccaccatgg atgatatggc attgcctcca ccaccccctg aactgctgtc
     2101 tgatcaacag aaggctggtt acggaggcag tcatatatca ggctatgcaa cgttgcggag
     2161 aggaccccct cctgctcccc ccaaaagaga ccagaacacc aagctctcca gagactggta
     2221 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2281 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2341 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2401 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2461 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2521 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2581 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2641 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2701 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2761 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2821 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2881 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2941 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3001 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3061 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3121 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3181 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3241 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3301 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3361 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3421 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3481 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3541 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3601 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3661 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3721 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3781 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3841 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3901 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3961 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4021 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4081 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4141 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4201 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4261 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4321 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4441 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4501 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4561 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4621 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4681 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4741 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4801 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4861 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4921 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4981 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5041 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5101 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5161 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5221 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5281 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5341 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5401 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5461 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5521 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5581 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5641 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5701 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5761 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5821 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5881 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5941 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6001 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6061 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6121 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6181 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6241 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6301 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6361 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6421 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6481 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6541 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6601 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6661 ctgacgtc
//
