LOCUS       sequence-398247-        6408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Tyr to Phe mutant FAKSPARK2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6408)
  AUTHORS   Li X, Combs JD 3rd, Salaita K, Shu X
  TITLE     Polarized focal adhesion kinase activity within a focal adhesion 
            during cell migration.
  JOURNAL   Nat Chem Biol. 2023 Jun 22. doi: 10.1038/s41589-023-01353-y.
  PUBMED    37349581
REFERENCE   2  (bases 1 to 6408)
  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: "Nat Chem 
            Biol. 2023 Jun 22. doi: 10.1038/s41589-023-01353-y."
COMMENT     Sequence Label: pCS2-mutFAKSPARK2
FEATURES             Location/Qualifiers
     source          1..6408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      91..100
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(175..177,178..180,181..891)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1087..1140
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1492..1494,1495..1497,1498..2208)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTFGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    2460..2594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2484..2503)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2538..2557
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2707..2727)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2707..2725)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2746..2762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2746..2762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2759..2781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2770..2786
                     /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(2794..2800,2801..2818,2819..2824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2839..2860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2977..2994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3148..3736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3228..3247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3907..4698,4699..4767))
                     /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     4530..4549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4768..4872)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4898..5353)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5035..5056)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5247..5266
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5424..6408
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccgctagc gccaccatgg agacggatga tttcgcggag
      121 ataatagatg agctcgagtc cggactcaga tctggcagcg gtggaggcga tatcatggtg
      181 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      241 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      301 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      361 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      421 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      481 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      541 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      601 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      661 aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      721 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      781 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      841 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggatccggc
      901 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggcag tgctggtggt
      961 agtgctggtg gtagtgctgg tggctctaga ggagaaatcg caaagagtct gaaagaaatt
     1021 gcaaaatcct tgaaggaaat agcgtggtca ctgaaagaaa ttgccaagtc cctcaaaggg
     1081 ggtaccgagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     1141 aagcttgcca ccatgtggta ttttggcaag atcaccagac gggagtcaga gcggttactg
     1201 ctcaatgcag agaacccgag agggaccttc ctcgtgcgag aaagtgagac cacgaaaggt
     1261 gcctactgcc tctcagtgtc tgacttcgac aacgccaagg gcctcaacgt gaagcactac
     1321 aagatccgca agctggacag cggcggcttc tacatcacct cccgcaccca gttcaacagc
     1381 ctgcagcagc tggtggccta ctactccaaa cacgccgatg gcctgtgcca ccgcctcacc
     1441 accgtgtgcc tcgagtccgg actcagatct ggcagcggtg gaggcgatat catggtgagc
     1501 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1561 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1681 accctgacct tcggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1741 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1801 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1921 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1981 gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2161 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg atccggcagt
     2221 gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggcagtgc tggtggtagt
     2281 gctggtggta gtgctggtgg ctctagaacc ttgagagaaa tcgaggaatt gttgaggaaa
     2341 atcattgaag acagcgtgcg cagtgtggcc gagctggagg acattgagaa gtggctgaag
     2401 aaaatctaag aattcaaggc ctctcgagcc tctagaacta tagtgagtcg tattacgtag
     2461 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2521 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2581 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2641 tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacccagctt
     2701 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2761 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     2821 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2881 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2941 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3001 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3061 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3121 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3181 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3241 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3301 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3361 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3421 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3481 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3541 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3601 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3661 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3721 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3781 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3841 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3901 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3961 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4021 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4081 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4141 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4201 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4261 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4321 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4381 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4441 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4501 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4561 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4621 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4681 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4741 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4801 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4861 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4921 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4981 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5041 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5101 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5161 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5221 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5281 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5341 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5401 cgggcctctt cgctattacg ccagtcgacc gccaattcaa tatggcgtat atggactcat
     5461 gccaattcaa tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt
     5521 gccaattcaa tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat
     5581 gccaattcaa tatggcggac ctggcactgt gccaactggg gaggggtcta cttggcacgg
     5641 tgccaagttt gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg
     5701 tgccaataat ggcggccata ttggctatat gccaggatca atatataggc aatatccaat
     5761 atggccctat gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca
     5821 tatagtattc catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc
     5881 ccatatacca tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa
     5941 tggtctctat atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg
     6001 gcgcctcccc cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac
     6061 gtcaatagga ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt
     6121 tctcacgccc cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat
     6181 ctattaatag taacttggca agtacattac tattggaagt acgccagggt acattggcag
     6241 tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat
     6301 tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt
     6361 aggcgtgcct aatgggaggt ctatataagc aatgctcgtt tagggaac
//
