LOCUS       sequence-248684-        3360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes PKA SPARK, a PKA activity reporter, as a Type 3 part to be 
            used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3360)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 3360)
  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"; doi: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: MTK3_029
FEATURES             Location/Qualifiers
     source          1..3360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     366..385
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(667..669,670..672,673..1383)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(712..733)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(973..992)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1320..1341
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1579..1632
                     /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"
     terminator      complement(2291..2325)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2396..3055)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2970..2989
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      join(3237..3360,1..465)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
       61 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      121 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      181 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      241 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      301 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      361 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      421 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      481 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      541 tcccctgatt ctgtggataa ccgtagtcgg tctcatatgg gcggtggagg gagcctgcgt
      601 cgcgccaccc tggtggacgg cggactcgag tccggactca gatctggcag cggtggaggc
      661 gatatcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      721 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      781 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      841 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      901 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      961 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1021 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1081 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1141 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1201 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1261 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1321 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1381 aagggatccg gcagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggc
     1441 agtgctggtg gtagtgctgg tggtagtgct ggtggctcta gaggagaaat cgcaaagagt
     1501 ctgaaagaaa ttgcaaaatc cttgaaggaa atagcgtggt cactgaaaga aattgccaag
     1561 tccctcaaag ggggtaccga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     1621 aatcctggcc caaagcttgc caccatgcat aagttttctc aagaacagat cggcgaaaac
     1681 attgtgtgca gggtcatttg taccacgggt caaattccca tccgagattt gtcagctgat
     1741 atttcacaag tgcttaagga aaaacgatcc ataaagaaag tttggacatt tggtagaaac
     1801 ccagcctgtg actatcattt aggaaacatt tcaagactgt caaataagca tttccaaata
     1861 ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg gacctggtta
     1921 aatgggcaaa aagtcgagaa gaacagcaat cagttactgt ctcaaggtga tgaaataacc
     1981 gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa cgacaaattt
     2041 aagcagtgcc tcgaacagaa caaagttgat cgcggatccg gcagtgctgg tggtagtgct
     2101 ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg tggtagtgct
     2161 ggtggctcta gaaccttgag agaaatcgag gaattgttga ggaaaatcat tgaagacagc
     2221 gtgcgcagtg tggccgagct ggaggacatt gagaagtggc tgaagaaaat cggatcctga
     2281 gaccagacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag
     2341 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg
     2401 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     2461 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     2521 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     2581 ttaaatcaaa actggtgaaa ctcacccagg gattggctga aacgaaaaac atattctcaa
     2641 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     2701 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     2761 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     2821 ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
     2881 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     2941 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     3001 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
     3061 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     3121 cattatggtg aaagttggaa cctcttacgt gcccgatcaa tcatgaccaa aatcccttaa
     3181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
//
