LOCUS       62056_20395        4140 bp DNA     circular SYN 17-NOV-2020
DEFINITION  Cloning vector pT-GTW6-CMV-mKate_2xmiR31ts3'UTR, complete sequence.
ACCESSION   MT891352
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4140)
  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 4140)
  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 4140)
  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..4140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    59..79
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     enhancer        151..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        455..658
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     protein_bind    689..713
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             748..765
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             766..1473
                     /label=PAmKate
                     /note="monomeric photoactivatable red fluorescent protein 
                     (Gunewardene et al., 2011)"
     misc_feature    1483..1524
                     /label=2xmiR31-TS
                     /note="2xmiR31-TS"
     protein_bind    complement(1544..1568)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1697..1818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1825..2280)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2307..2411
                     /label=AmpR promoter
     CDS             2412..3269
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      3443..4031
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 catttcaggt gatccagtta tttaatagtc aattacggca tatatctagc agtgatatca
       61 actttgtata gaaaagttgg ctccgaattc tagttattaa tagtaatcaa ttacggggtc
      121 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      181 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      481 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      541 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      601 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      661 tttagtgaac cgtcagatcg aattcgaccc aagtttgtac aaaaaagcag gctgattctt
      721 ccatttcagg tggatcccgc caccatgcac catcaccatc accatgtgtc taagggcgaa
      781 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac
      841 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
      901 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc
      961 atgtacggca gcaaaacctt catcaaccac acccagggca tccccgactt ctttaagcag
     1021 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     1081 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     1141 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1201 tccaccgaga tgctgtaccc cgctgacggc ggcctggaag gcagaagcga catggccctg
     1261 aagctcgtgg gcgggggcca cctgatctgc aacttgaaga ccacatacag atccaagaaa
     1321 cccgctaaga acctcaagat gcccggcgtc tactatgtgg acagaagact ggaaagaatc
     1381 aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc
     1441 gacctcccta gcaaactggg gcacaaactt aattgaaagc ttagctatgc cagcatcttg
     1501 cctagctatg ccagcatctt gcctttaatt aataaagcta gttacccagc tttcttgtac
     1561 aaagtggtac gcgtgacggc cgcgactcta gatcataatc agccatacca catttgtaga
     1621 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1681 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1801 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     1861 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1921 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1981 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2041 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2101 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2161 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2221 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2281 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2341 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2401 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2461 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2521 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2581 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2641 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2701 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2761 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2821 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2881 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2941 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3001 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3061 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3121 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3181 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3241 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3301 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3361 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3421 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3481 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3541 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3601 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3661 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3721 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3781 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3841 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3901 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3961 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4021 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4081 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
//
