LOCUS       sequence-249777-        2491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a nuclear localized EBFP2 coexpression cassette as a Type 4a
            part to be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2491)
  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 2491)
  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: MTK4a_017
FEATURES             Location/Qualifiers
     source          1..2491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      56..644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     545..564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             768..824
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             825..845
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(861..863,864..1577)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIKV
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLLE
                     FRTAAGITLGMDELYK"
     primer_bind     complement(903..924)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     terminator      complement(1601..1635)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(1706..2365)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2280..2299
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
       61 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
      121 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
      181 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
      241 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
      301 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      361 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
      421 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
      481 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
      541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
      601 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
      661 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
      721 cccctgattc tgtggataac cgtagtcggt ctcaatccgg aagcggagct actaacttca
      781 gcctgctgaa gcaggctggt gacgtggagg agaaccctgg acctcccaag aagaagagga
      841 aggtggagga cgccggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      901 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgagg ggcgagggcg
      961 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1021 ccgtgccctg gcccaccctc gtgaccaccc tgagccacgg cgtgcagtgc ttcgcccgct
     1081 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1141 aggagcgcac catcttcttc aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt
     1201 tcgagggcga caccctggtg aaccgcatcg agctgaaggg cgtcgacttc aaggaggacg
     1261 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacatc tatatcatgg
     1321 ccgtcaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac gtggaggacg
     1381 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1441 tgctgcccga cagccactac ctgagcaccc agtccgtgct gagcaaagac cccaacgaga
     1501 agcgcgatca catggtcctg ctggagttcc gcaccgccgc cgggatcact ctcggcatgg
     1561 acgagctgta caagtaactc gagtggctga gaccagacca ataaaaaacg cccggcggca
     1621 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     1681 gagtccaagc gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt
     1741 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     1801 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
     1861 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     1921 gattggctga aacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     1981 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     2041 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     2101 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag
     2161 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     2221 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     2281 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     2341 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
     2401 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     2461 gcccgatcaa tcatgaccaa aatcccttaa c
//
