LOCUS       sequence-388856-        6686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RAPH1 fragment (868-1062) tagged to eGFP in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6686)
  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 6686)
  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-F3
FEATURES             Location/Qualifiers
     source          1..6686
                     /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(2277..2294)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2283..2507
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2553..2981
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2640..2659)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2850..2871
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2990..3010)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2995..3324
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3175..3310
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3237..3256
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3391..4185
                     /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(3445..3464)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4055..4074
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4359..4480
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4396..4415)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4450..4469
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4529..4545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4529..4545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4542..4564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4553..4569
                     /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(4577..4583,4584..4601,4602..4607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4622..4643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4760..4777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4931..5519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5011..5030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5690..6481,6482..6550))
                     /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     6313..6332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6551..6655)
                     /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 atcctttcca ccccctccaa ctccccctgc
     1681 catggaatct cagcccttaa agcctgtccc agcaaatgta gctccacagt cccctcctgc
     1741 agtaaaagca aagcccaagt ggcagcccag ctccatccca gtcccttctc cggacttccc
     1801 tcctccccct cctgaaagca gcctggtgtt tcctcctcca cccccatcac ctgtcccagc
     1861 cccaccacca ccacctccac ccacagcttc tcctacccct gacaaaagtg gatctccagg
     1921 caaaaagacc agtaagacgt ccagccctgg gggaaagaaa ccacccccaa ccccacagcg
     1981 caactccagc attaaatcca gcagtggtgc agagcacccc gagcccaaga gaccctcggt
     2041 ggacagtcta gtcagcaagt ttacaccgcc agcagaatca gggtctccca gcaaggagac
     2101 cctaccacct cctgcagcac cccccaagcc tggaaaactc aatctttctg gagtcaacct
     2161 tcctggagtt ctccaacaag ggtgtgtgtc agcaaaagcc cctgttctga gtgggcgtgg
     2221 aaaggactcc gtggtgtagc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2281 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2341 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2401 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2461 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2521 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2581 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2641 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2701 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2761 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2821 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2881 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2941 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3001 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3061 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3121 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3181 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3241 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3301 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3361 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3421 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3481 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3541 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3601 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3661 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3721 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3781 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3841 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3901 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3961 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4021 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4081 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4141 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4201 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4261 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4321 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4381 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4441 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4501 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4561 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4621 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4681 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4741 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4801 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4861 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4921 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5101 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5341 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5521 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5581 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5641 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5701 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5761 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5821 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5881 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5941 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6001 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6061 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6121 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6181 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6241 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6301 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6361 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6421 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6481 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6541 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6601 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6661 ttccccgaaa agtgccacct gacgtc
//
