LOCUS       62056_3860        4596 bp DNA     circular SYN 17-NOV-2020
DEFINITION  Cloning vector pBI-CMV-EGFP-mKate-3xmiR31ts3'UTR, complete sequence.
ACCESSION   MT891347
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4596)
  AUTHORS   Frei T, Cella F, Tedeschi F, Gutierrez J, Stan GB, Khammash M, 
            Siciliano V.
  TITLE     Characterization and mitigation of gene expression burden in 
            mammalian cells
  JOURNAL   Nat Commun 11 (1), 4641 (2020)
  PUBMED    32934213
REFERENCE   2  (bases 1 to 4596)
  AUTHORS   Frei T, Cella F, Tedeschi F, Gutierrez J, Stan G-B., Khammash M, 
            Siciliano V.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-AUG-2020) Synthetic and Systems Biology for 
            Biomedicine, Italian Institute of Technology-IIT, Largo Barsanti e 
            Martteucci 53, Naples, Campania 80125, Italy
REFERENCE   3  (bases 1 to 4596)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun"; date: "2020"; volume: "11"; issue: "1"; pages: "4641"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-AUG-2020) Synthetic and Systems Biology for Biomedicine, Italian
            Institute of Technology-IIT, Largo Barsanti e Martteucci 53, Naples,
            Campania 80125, Italy"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..4596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    293..374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(556..1144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1318..2175)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2176..2280)
                     /label=AmpR promoter
     polyA_signal    complement(2313..2394)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2514..3230)
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     enhancer        3389..3692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3693..3896
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             3940..3957
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             join(3958..4596,1..69)
                     /codon_start=1
                     /label=PAmKate
                     /note="monomeric photoactivatable red fluorescent protein 
                     (Gunewardene et al., 2011)"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
ORIGIN
        1 gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac
       61 aaacttaatt gaaagcttag ctatgccagc atcttgccta gctatgccag catcttgcct
      121 agctatgcca gcatcttgcc tttaattaaa gctagttacc caagcttgtc gacgatatct
      181 ccagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
      241 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
      301 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
      361 atttttttca ctgccccgag cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      421 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      481 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1321 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1381 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1441 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1501 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1561 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1621 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1681 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1741 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1801 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1861 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1921 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1981 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2041 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2101 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2161 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2281 cacatttccc cgaaaagtgc cacctgacgt cggcagtgaa aaaaatgctt tatttgtgaa
     2341 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     2401 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     2461 aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctagactg cagcttgtac
     2521 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     2581 tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg
     2641 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     2701 acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc
     2761 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
     2821 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     2881 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     2941 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     3001 tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag
     3061 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     3121 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     3181 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcaccg
     3241 gtgaattctc caggcgatct gacggttcac taaacgagct ctgcttatat aggcctccca
     3301 ccgtacacgc cacctcgaca tactcgagta gttattaata gtaatcaatt acggggtcat
     3361 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3421 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     3481 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     3541 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     3601 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     3661 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     3721 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     3781 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     3841 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
     3901 tagtgaaccg tcagatccgc tagggatccc gccaccatgc accatcacca tcaccatgtg
     3961 tctaagggcg aagagctgat taaggagaac atgcacatga agctgtacat ggagggcacc
     4021 gtgaacaacc accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
     4081 cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg
     4141 gctaccagct tcatgtacgg cagcaaaacc ttcatcaacc acacccaggg catccccgac
     4201 ttctttaagc agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
     4261 gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
     4321 gtcaagatca gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc
     4381 ggctgggagg cctccaccga gatgctgtac cccgctgacg gcggcctgga aggcagaagc
     4441 gacatggccc tgaagctcgt gggcgggggc cacctgatct gcaacttgaa gaccacatac
     4501 agatccaaga aacccgctaa gaacctcaag atgcccggcg tctactatgt ggacagaaga
     4561 ctggaaagaa tcaaggaggc cgacaaagag acctac
//
