LOCUS       sequence-388859-        5977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the RAPH1 fragment (535-868) tagged to GST in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5977)
  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 5977)
  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: GST-RAPH1-F2
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             936..956
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2027..2049)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2194..2212)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2280..2384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2385..2453,2454..3245)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2603..2622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3416..4004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3905..3924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4158..4175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4248..4325
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4326..5408
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4345..4364)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5457..5462,5463..5480,5481..5487)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5495..5511
                     /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)."
     primer_bind     5519..5535
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5519..5535
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5531..5977,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5551..5568)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5551..5567)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5560..5582)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccgaaaa tctatatttt caatcaggat
      961 cgggttccag ttcttccagc atcccagagt ctcagtcaaa ccactccaat cagtctgata
     1021 gcggagtttc tgacacccag ccagcaggac acgtccgttc ccagagcatt gtgagctccg
     1081 tattctctga agcctggaaa cgaggcactc agttggaaga gtccagcaag gccagaatgg
     1141 agtctatgaa tcggccctac acttcacttg tgcccccttt atccccgcaa cctaagatag
     1201 tcacccccta cactgcttca cagccttcac cacctctacc tcctccgcca cccccacctc
     1261 ctcctccacc accccctcca ccaccccctc ctcccccact ccccagccag tctgcacctt
     1321 ctgcaggctc agcagcccca atgttcgtca agtacagcac aataacacgg ctacagaatg
     1381 cgtctcagca ttcaggggcc ctgtttaagc cgccaacacc cccagtgatg cagtcacagt
     1441 cagtgaagcc tcagatcctg gtacccccca atggagttgt tccaccaccc cctccccctc
     1501 ctccaccccc aaccccaggc tctgccatgg cccagctaaa gcctgcaccg tgtgccccat
     1561 cccttccaca gttcagtgcc ccgcctcctc cactgaagat ccatcaagtt cagcatatta
     1621 ctcaggtggc tcccccaaca ccccccccac ctcctcctat ccctgcaccc ctccctcccc
     1681 aagctccccc aaaacccctt gtgaccatcc ccgcaccaac cagcaccaag actgtggcac
     1741 ctgttgtgac tcaagctgca ccacccacac ctactcctcc agtgccccca gcaaaaaagc
     1801 agccagcttt ccctgcttct tacattccac cctctccccc tacccctcct gttccagtac
     1861 ccccgccaac attacccaag caacagagct tctgtgcaaa accccctccc tctccactgt
     1921 caccggtgcc ctcggtcgtg aagcagatag ccagccagta gctcgagcgg ccgcatcgtg
     1981 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2041 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2101 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     2161 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2221 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2281 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2341 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2401 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2461 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2521 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2581 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2641 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2701 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2761 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2821 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2881 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2941 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3001 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3061 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3121 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3181 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3241 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3301 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3361 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3421 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3481 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3541 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3601 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3661 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3721 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3781 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3841 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3901 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3961 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4021 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4081 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4141 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4201 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     4261 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4321 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4381 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4441 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4501 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4561 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4621 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4681 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4741 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4801 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4861 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4921 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4981 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5041 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5101 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5161 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5221 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5281 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5341 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5401 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     5461 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5521 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     5581 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5641 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5701 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     5761 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     5821 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     5881 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     5941 aggccagacg cgaattattt ttgatggcgt tggaatt
//
