LOCUS       EF1a-VP16-ZF37-2        4950 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4950)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene 8.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4950
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     terminator      555..641
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      733..760
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        828..2009
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1058..2000
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     regulatory      2071..2080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2080..2145
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2152..2172
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2209..2442
                     /label=VP16
     misc_feature    2446..2715
                     /label=ZF37
                     /note="ZF37 domain from COMET"
     CDS             2725..2781
                     /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             2794..3495
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3507..3731
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3754..3777
                     /label=pENTR2B.Seq.REV
     CDS             3853..4662
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     misc_feature    join(4736..4950,1..405)
                     /label=pBR322_Origin
     rep_origin      join(4752..4950,1..390)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
       61 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      121 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      181 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      241 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      301 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      361 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      421 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      481 gataaccgta ttaccgctag catggatctc ggggacgtct aactactaag cgagagtagg
      541 gaactgccag gcatcaaata aaacgaaagg ctcagtcgga agactgggcc tttcgtttta
      601 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
      661 acgttgtgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
      721 atcaaactaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc
      781 ctgttagtta gttacttaag ctcgggccca cctgcagtct acttactgct ccggtgcccg
      841 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      901 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      961 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     1021 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     1081 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacgcccct
     1141 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     1201 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cttgggcgct
     1261 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     1321 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     1381 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     1441 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     1501 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     1561 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1621 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1681 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1741 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1801 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1861 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1921 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1981 gtttttttct tccatttcag gtgtcgtgag agctcgttta gtgaaccgtc agatcgcctg
     2041 gagacgccat ccacgctgta atgagccacc gccaccatgg attataagga tcacgatggc
     2101 gactacaagg accacgatat tgactacaaa gacgatgacg ataaaatggc tcctaagaaa
     2161 aagcgcaaag tcggtatcca tggcgttccc gtcgacggtg gactgcaggc cccaccaacc
     2221 gatgtttctt taggtgatga gttacatttg gacggtgagg acgtagctat ggcacatgca
     2281 gatgccttag acgattttga cttggatatg ttaggagatg gagattcacc aggacctggt
     2341 ttcacaccac acgattcagc tccatacggt gctttggaca tggccgattt cgaatttgaa
     2401 cagatgttta ctgatgcctt gggtatagac gaatacggtg gtggagctag acccggggag
     2461 cgccccttcc agtgtcgcat ttgcatgcgg aacttttcga gaaatttcat tttgcaaaga
     2521 catacccgta ctcataccgg tgaaaaaccg tttcagtgtc ggatctgtat gcgaaatttc
     2581 tccgatagag ctaatttgag gaggcatctg cgtacgcaca ccggcgagaa gccattccaa
     2641 tgccgaatat gcatgcgcaa cttcagtaga catgatcaat tgactagaca cctaaaaacc
     2701 cacctgagag gatctggaag cggagctact aacttcagcc tgctgaagca ggctggagac
     2761 gtggaggaga accctggacc tgcaccagga agtatgagcg agctgattaa ggagaacatg
     2821 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     2881 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     2941 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     3001 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     3061 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     3121 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     3181 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
     3241 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     3301 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     3361 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgaaacg
     3421 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     3481 cacaagctta attaacaacg ttgcacctgt gccttctagt tgccagccat ctgttgtttg
     3541 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3601 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3661 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3721 gggctctatg gcgctcgctg actgcaggtg cccgtgtctc aaaatctctg atgttacatt
     3781 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     3841 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     3901 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     3961 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     4021 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     4081 atgcctcttc cgaccatcaa gcattttatc cgtactcctg gtgatgcatg gttactcacc
     4141 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     4201 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     4261 tgtcctttta acagcgatcg cgtatttcgt ctagctcagg cgcaatcacg aatgaataac
     4321 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     4381 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     4441 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     4501 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     4561 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     4621 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     4681 ttgtaacatt attcagattg ggccccgttc cactgagcgt cagaccccgt agaaaagatc
     4741 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4801 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4861 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4921 ggccaccact tcaagaactc tgtagcaccg
//
