LOCUS       sequence-251627-        2494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a nuclear localized EBFP2 (T2A) 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 2494)
  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 2494)
  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_009
FEATURES             Location/Qualifiers
     source          1..2494
                     /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..821
                     /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             828..848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(864..866,867..1580)
                     /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(906..927)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     terminator      complement(1604..1638)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(1709..2368)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2283..2302
                     /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 aagcggagag ggcagaggaa
      781 gtctgctaac atgcggtgac gtcgaggaga atcctggacc tggatccccc aagaagaaga
      841 ggaaggtgga ggacgccgga tccgtgagca agggcgagga gctgttcacc ggggtggtgc
      901 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg aggggcgagg
      961 gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1021 tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca cggcgtgcag tgcttcgccc
     1081 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1141 tccaggagcg caccatcttc ttcaaggacg acggcaccta caagacccgc gccgaggtga
     1201 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcgtcgac ttcaaggagg
     1261 acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaac atctatatca
     1321 tggccgtcaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacgtggagg
     1381 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1441 tgctgctgcc cgacagccac tacctgagca cccagtccgt gctgagcaaa gaccccaacg
     1501 agaagcgcga tcacatggtc ctgctggagt tccgcaccgc cgccgggatc actctcggca
     1561 tggacgagct gtacaagtaa ctcgagtggc tgagaccaga ccaataaaaa acgcccggcg
     1621 gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa
     1681 caggagtcca agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact
     1741 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     1801 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     1861 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     1921 agggattggc tgaaacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     1981 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     2041 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     2101 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacga aattccggat
     2161 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     2221 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     2281 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     2341 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     2401 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     2461 cgtgcccgat caatcatgac caaaatccct taac
//
