LOCUS       sequence-249805-        3671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes tagBFP, thosea asigna virus 2A (T2A) and Blasticidin 
            resistance as a multicistronic cassette to be used in the MTK 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3671)
  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 3671)
  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: MTK6_004
FEATURES             Location/Qualifiers
     source          1..3671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      128..716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     617..636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1162..1365
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1316..1336
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1387..2085
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2086..2106
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2137..2190
                     /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             2197..2595
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2607..2624)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2613..2820
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      complement(2853..2887)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2958..3617)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3532..3551
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tgatcttatt tcattatggt gaaagttgga acctcttacg tgcccgatca atcatgacca
       61 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      121 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      181 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      241 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      301 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      361 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      421 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      481 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      541 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      601 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      661 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
      721 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
      781 ttcctgcgtt atcccctgat tctgtggata accgtagtcg gtctcataca gcggtgggct
      841 ctatggggta cctacgtagg tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      901 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      961 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1021 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1081 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1141 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     1201 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1261 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     1321 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
     1381 cagataatga gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc
     1441 gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
     1501 cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg
     1561 gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac
     1621 ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
     1681 gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
     1741 gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc
     1801 ggctgggagg ccttcaccga aacgctgtac cccgctgacg gcggcctgga aggcagaaac
     1861 gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat
     1921 agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga
     1981 ctggaaagaa tcaaggaggc caacaacgaa acctacgtcg agcagcacga ggtggcagtg
     2041 gccagatact gcgacctccc tagcaaactg gggcacaagc ttaatcccaa gaagaagagg
     2101 aaggtggagg acgcccgtac gagtactgga agcggagagg gcagaggaag tctgctaaca
     2161 tgcggtgacg tcgaggagaa tcctggacct ggatccatgg ccaagccttt gtctcaagaa
     2221 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     2281 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     2341 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     2401 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     2461 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     2521 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     2581 gtgtgggagg gctaactcga gcgacgcctc gactgtgcct tctagttgcc agccatctgt
     2641 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2701 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2761 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2821 tgcggtgggc tctatgagtt gagaccagac caataaaaaa cgcccggcgg caaccgagcg
     2881 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     2941 gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     3001 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     3061 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     3121 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     3181 gaaacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     3241 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     3301 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     3361 tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc
     3421 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     3481 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     3541 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     3601 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     3661 acgcccggta g
//
