LOCUS       62056_41        8543 bp DNA     circular SYN 17-NOV-2020
DEFINITION  Cloning vector (220)_P_EF1a-mCitrine-T, complete sequence.
ACCESSION   MT891332
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8543)
  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 8543)
  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 8543)
  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..8543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..266
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     polyA_signal    283..490
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        526..1707
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1729..2445
                     /label=mEYFP
                     /note="enhanced YFP with monomerizing A206K mutation
                     (Zacharias et al., 2002)"
     misc_binding    2459..2480
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     misc_binding    2481..2502
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     misc_binding    2503..2524
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     regulatory      complement(2561..2579)
                     /label=SP6 promoter
                     /note="SP6 promoter"
                     /regulatory_class="promoter"
     polyA_signal    2740..2861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3000..3265
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     polyA_signal    3282..3506
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3527..4709
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     misc_feature    4718..4727
                     /label=Kozak sequence
                     /note="Kozak sequence"
     ncRNA           5491..5619
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the
                     human miR-30a microRNA (Zeng et al., 2002)"
     misc_binding    5828..5849
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     misc_binding    5850..5871
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     misc_binding    5872..5893
                     /label=miR-FF5 binding site
                     /bound_moiety="miR-FF5"
                     /note="mir FF5 target"
     promoter        complement(5931..5949)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     regulatory      6110..6231
                     /label=SV40 poly(A) signal
                     /note="SV40 poly(A) signal"
                     /regulatory_class="polyA_signal_sequence"
     regulatory      6321..6610
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     rep_origin      complement(6698..7286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7460..8317)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8318..8422)
                     /label=AmpR promoter
ORIGIN
        1 gtcttctcca tcggatcctg atcaggggag ctcacgggga cagccccccc ccaaagcccc
       61 cagggatgta attacgtccc tcccccgcta gggggcagca gcgaccgccc ggggctccgc
      121 tccggtccgg cgctcccccc gcatcccgag ccggcagcgt gcggggacag cccgggcacg
      181 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
      241 acacctgggg gatacgggga aaaccgcggc atatggcctc gactgtgcct tctagttgcc
      301 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      361 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
      421 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
      481 atgctgggga tgcggtgggc tctatggctt ctgagggcta gcgatgctcc ggtgcccgtc
      541 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
      601 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
      661 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
      721 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
      781 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg
      841 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
      901 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
      961 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     1021 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1081 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1141 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1201 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1261 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1321 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1381 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1441 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     1501 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     1561 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     1621 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     1681 ttttttcttc catttcaggt gtcgtgacat aaactgccct agaccaccat ggtgagcaag
     1741 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1801 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1861 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1921 ttcggctacg gcctgatgtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1981 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2041 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2101 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2161 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2221 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2281 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2341 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2401 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaatg actccggaaa
     2461 gcactctgat ttgacaatta aagcactctg atttgacaat taaagcactc tgatttgaca
     2521 attaactagt atgtacaagt agtaatctag agggccctat tctatagtgt cacctaaatg
     2581 ctagagctcg ctgatcagtc catagtcgac ggatccaccg gatctagata actgatcata
     2641 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     2701 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     2761 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2821 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     2881 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     2941 ggccggtaag tcaatgatag cactcgatgc cgcatagtta agccagtatc tgctccctgg
     3001 tcttctccat cggatcctga tcaggggagc tcacggggac agcccccccc caaagccccc
     3061 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgaccgcccg gggctccgct
     3121 ccggtccggc gctccccccg catcccgagc cggcagcgtg cggggacagc ccgggcacgg
     3181 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     3241 cacctggggg atacggggaa aaccgcggca tatggcctcg actgtgcctt ctagttgcca
     3301 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3361 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3421 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3481 tgctggggat gcggtgggct ctatggcttc tgagggctag cgatatcgct ccggtgcccg
     3541 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     3601 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     3661 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     3721 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     3781 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacgcccct
     3841 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     3901 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
     3961 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     4021 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     4081 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     4141 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     4201 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     4261 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     4321 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     4381 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     4441 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     4501 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     4561 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     4621 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     4681 gtttttttct tccatttcag gtgtcgtgag cggccgcgcc accatggtgt ctaagggcga
     4741 agagctgatc aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca
     4801 ccaattcaaa tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag
     4861 gatcaaagtc atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt
     4921 catgtatggc agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca
     4981 gtcctttcct gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt
     5041 caccgtcacg caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag
     5101 aggggtaaac tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc
     5161 taatacagag atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact
     5221 gaaagttgat ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtgagtatg
     5281 tgctcgcttc ggcagcacat atactatgtc gaatgaggct tcagtacttt acagaatcgt
     5341 tgcctgcaca tcttggaaac acttgctggg attacttctt caggttaacc caacagaagg
     5401 tacgtatgct gttgacagtg agcgccgctt gaagttttta attaaatagt gaagccacag
     5461 atgtatttaa ttaaagactt caagcggtgc ctactgcctc ggagaattca aggggctact
     5521 ttaggagcaa ttatcttgtt tactaaaact gaataccttg ctatctcttt gatacatttt
     5581 tacaaagctg aattaaaatg gtataaatta aatcactttt ttcaattgtt tccttttttt
     5641 tcctcaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtgtccat gccgttgatc
     5701 accgcctgga aaggatcgag gagagtgaca atgaaaccta cgtagtgcaa agagaagtgg
     5761 cagttgccaa atacagcaac cttggtggtg gcatggacga gctgtacaag tagtaacatg
     5821 cttcgaaaag cactctgatt tgacaattaa agcactctga tttgacaatt aaagcactct
     5881 gatttgacaa ttaggcgcgc ctgtacaagt agtaatctag agggccctat tctatagtgt
     5941 cacctaaatg ctagagctcg ctgatcagtc catagtcgac ggatccaccg gatctagata
     6001 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     6061 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     6121 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6181 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa
     6241 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6301 ttaaccaata ggccgaaatc ggggagctca cggggacagc ccccccccaa agcccccagg
     6361 gatgtaatta cgtccctccc ccgctagggg gcagcagcga ccgcccgggg ctccgctccg
     6421 gtccggcgct ccccccgcat cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
     6481 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     6541 ctgggggata cggggaaaat gatcaagatc ttctacactg ggacgcaagg attatcctcg
     6601 aggcgcgcga agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6661 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6721 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6781 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6841 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6901 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6961 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7021 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7081 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     7141 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7201 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7261 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7321 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7381 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7441 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7501 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7561 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7621 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7681 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7741 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7801 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7861 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7921 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7981 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8041 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     8101 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8161 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8221 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8281 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8341 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8401 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     8461 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     8521 gttaagccag tatctgctcc ctg
//
