LOCUS       sequence-388855-        7103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RAPH1 fragment (535-868) tagged to eGFP in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7103)
  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 7103)
  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-F2
FEATURES             Location/Qualifiers
     source          1..7103
                     /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(2694..2711)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2700..2924
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2970..3398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3057..3076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3267..3288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3407..3427)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3412..3741
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3592..3727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3654..3673
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3808..4602
                     /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(3862..3881)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4472..4491
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4776..4897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4813..4832)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4867..4886
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4946..4962)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4946..4962)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4959..4981)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4970..4986
                     /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(4994..5000,5001..5018,5019..5024))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5039..5060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5177..5194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5348..5936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5428..5447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6107..6898,6899..6967))
                     /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     6730..6749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6968..7072)
                     /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 atcctcgggt tccagttctt ccagcatccc
     1681 agagtctcag tcaaaccact ccaatcagtc tgatagcgga gtttctgaca cccagccagc
     1741 aggacacgtc cgttcccaga gcattgtgag ctccgtattc tctgaagcct ggaaacgagg
     1801 cactcagttg gaagagtcca gcaaggccag aatggagtct atgaatcggc cctacacttc
     1861 acttgtgccc cctttatccc cgcaacctaa gatagtcacc ccctacactg cttcacagcc
     1921 ttcaccacct ctacctcctc cgccaccccc acctcctcct ccaccacccc ctccaccacc
     1981 ccctcctccc ccactcccca gccagtctgc accttctgca ggctcagcag ccccaatgtt
     2041 cgtcaagtac agcacaataa cacggctaca gaatgcgtct cagcattcag gggccctgtt
     2101 taagccgcca acacccccag tgatgcagtc acagtcagtg aagcctcaga tcctggtacc
     2161 ccccaatgga gttgttccac caccccctcc ccctcctcca cccccaaccc caggctctgc
     2221 catggcccag ctaaagcctg caccgtgtgc cccatccctt ccacagttca gtgccccgcc
     2281 tcctccactg aagatccatc aagttcagca tattactcag gtggctcccc caacaccccc
     2341 cccacctcct cctatccctg cacccctccc tccccaagct cccccaaaac cccttgtgac
     2401 catccccgca ccaaccagca ccaagactgt ggcacctgtt gtgactcaag ctgcaccacc
     2461 cacacctact cctccagtgc ccccagcaaa aaagcagcca gctttccctg cttcttacat
     2521 tccaccctct ccccctaccc ctcctgttcc agtacccccg ccaacattac ccaagcaaca
     2581 gagcttctgt gcaaaacccc ctccctctcc actgtcaccg gtgccctcgg tcgtgaagca
     2641 gatagccagc cagtagctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2701 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2761 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2821 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2881 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2941 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3001 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3061 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3121 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3181 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3241 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3301 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3361 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3421 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3481 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3601 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3661 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3721 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3781 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3841 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3901 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3961 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4021 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4081 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4141 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4201 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4261 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4321 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4381 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4441 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4501 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4561 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4621 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4681 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4741 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4801 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4861 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4921 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4981 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5041 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5101 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5161 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5221 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5281 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5341 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5401 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5461 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5521 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5581 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5641 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5701 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5761 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5821 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5881 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5941 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6001 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6061 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6121 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6181 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6241 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6301 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6361 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6421 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6481 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6541 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6601 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6661 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6721 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6781 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6841 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6901 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6961 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7021 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7081 cccgaaaagt gccacctgac gtc
//
