LOCUS       62056_31        9978 bp DNA     circular SYN 17-NOV-2020
DEFINITION  Cloning vector (218)_P_TRE-(HDV)mCitri, complete sequence.
ACCESSION   MT891330
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9978)
  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 9978)
  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 9978)
  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..9978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..35
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac left (5') inverted repeat"
     misc_feature    361..385
                     /label=ConS scar
                     /note="ConS scar"
     misc_feature    390..414
                     /label=bidirectional promoter artefact
                     /note="bidirectional promoter artefact"
     promoter        415..729
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     misc_feature    767..919
                     /label=HDV
                     /note="HDV"
     CDS             917..1630
                     /label=mEYFP
                     /note="enhanced YFP with monomerizing A206K mutation
                     (Zacharias et al., 2002)"
     regulatory      1668..1789
                     /label=SV40 poly(A) signal
                     /note="SV40 poly(A) signal"
                     /regulatory_class="polyA_signal_sequence"
     misc_feature    1891..1915
                     /label=Con1 scar
                     /note="Con1 scar"
     regulatory      1954..2219
                     /note="misc_signal; INS"
                     /regulatory_class="other"
     polyA_signal    2236..2460
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2461..3642
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             3665..4408
                     /label=tTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
     CDS             4484..5194
                     /label=mRuby3
                     /note="monomeric red fluorescent protein derived from
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
     polyA_signal    complement(5232..5353)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    5455..5479
                     /label=ConE scar
                     /note="ConE scar"
     promoter        5499..5828
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        5876..5923
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             5942..6538
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     CDS             6545..7537
                     /label=Delta-TK
                     /note="truncated herpes simplex virus thymidine kinase
                     (Salomon et al., 1995)"
     promoter        complement(7567..7585)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    7746..7867
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   8064..8126
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac right (3') inverted repeat"
     regulatory      complement(8175..8201)
                     /label=KanR terminator
                     /note="KanR terminator"
                     /regulatory_class="terminator"
     CDS             complement(8205..9017)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     regulatory      complement(9018..9165)
                     /label=KanR promoter
                     /note="KanR promoter"
                     /regulatory_class="promoter"
     rep_origin      9242..9830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
       61 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
      121 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
      181 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
      241 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
      301 atagatatca actagaatgc tagacctttc gtcttcaaga accctgaatt cgcatctaga
      361 ctgaactggc cgataattgc agacgaacgc cgtacacgcc acctcgacat actcgagttt
      421 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      481 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      541 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      601 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      661 tgtcgaggta ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt
      721 cagatcgcct ggagatttcg agctcggtgc ggccgcgcca cctatgggaa tggcgggacc
      781 ccactctgca gggtccgcgt tccatccttt cttacctgat ggccggcatg gtcccagcct
      841 cctcgctggc gccggctggg caacattccg aggggaccgt cccctcggta atggcgaatg
      901 ggacgcacaa atctctctaa gcaagggcga ggagctgttc accggggtgg tgcccatcct
      961 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1021 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1081 gccctggccc accctcgtga ccaccttcgg ctacggcctg atgtgcttcg cccgctaccc
     1141 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1201 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1261 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1321 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1381 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1441 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1501 gcccgacaac cactacctga gctaccagtc caagctgagc aaagacccca acgagaagcg
     1561 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1621 gctgtacaag ggatcctaac tcgagcggcc gcttcgagca gacatgataa gatacattga
     1681 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1741 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1801 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta
     1861 aaacctctac aaatgtggta aaatcggctg ccaaaccaga tgtcaacaca gctacaacgg
     1921 atgccgcata gttaagccag tatctgctcc ctggtcttct ccatcggatc ctgatcaggg
     1981 gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg
     2041 ctagggggca gcagcgaccg cccggggctc cgctccggtc cggcgctccc cccgcatccc
     2101 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     2161 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg ggggatacgg ggaaaaccgc
     2221 ggcatatggc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2281 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2341 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2401 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2461 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2521 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2581 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2641 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     2701 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     2761 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     2821 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     2881 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     2941 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     3001 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     3061 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     3121 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     3181 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     3241 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     3301 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     3361 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     3421 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     3481 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     3541 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     3601 acagtggttc aaagtttttt tcttccattt caggtgtcgt gacataaact gccctagacc
     3661 acctatgtct agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga
     3721 ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc
     3781 tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat
     3841 gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt
     3901 acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt
     3961 acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt
     4021 tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca
     4081 ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga
     4141 aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca
     4201 ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa
     4261 acaacttaaa tgtgaaagtg ggtccccggc cgacgccctg gacgacttcg acctggacat
     4321 gctgccggcc gacgccctgg acgacttcga cctggacatg ctgccggccg acgccctgga
     4381 cgacttcgac ctggacatgc tgccggggca accaggttct ggcgcgacca actttagcct
     4441 gctgaaacag gcgggcgatg tggaagaaaa ccctggaccg gtcatggtgt ctaagggcga
     4501 agagctgatc aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca
     4561 ccaattcaaa tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag
     4621 gatcaaagtc atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt
     4681 catgtatggc agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca
     4741 gtcctttcct gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt
     4801 caccgtcacg caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag
     4861 aggggtaaac tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc
     4921 taatacagag atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact
     4981 gaaagttgat ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa
     5041 gaccgtcggg aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat
     5101 cgaggagagt gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag
     5161 caaccttggt ggtggcatgg acgagctgta caagggatcc taactcgagc ggccgcttcg
     5221 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     5281 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     5341 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     5401 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg gctgagcagt
     5461 tacagagatg ttacgaacca ctagtgcact gcagtacagt gtgtcagtta gggtgtggaa
     5521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5581 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5641 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     5701 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     5761 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     5821 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga
     5881 caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac
     5941 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     6001 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     6061 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     6121 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     6181 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     6241 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     6301 gcccgcgtgg ttcctggcca ccgtcggcgt ttcgcccgac caccagggca agggtctggg
     6361 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     6421 ggaaacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     6481 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg
     6541 atccatgccc acgctactgc gggtttatat agacggtcct cacgggatgg ggaaaaccac
     6601 caccacgcaa ctgctggtgg ccctgggttc gcgcgacgat atcgtctacg tacccgagcc
     6661 gatgacttac tggcaggtgc tgggggcttc cgagacaatc gcgaacatct acaccacaca
     6721 acaccgcctc gaccagggtg agatatcggc cggggacgcg gcggtggtaa tgacaagcgc
     6781 ccagataaca atgggcatgc cttatgccgt gaccgacgcc gttctggctc ctcatatcgg
     6841 gggggaggct gggagctcac atgccccgcc cccggccctc accctcatct tcgaccgcca
     6901 tcccatcgcc gccctcctgt gctacccggc cgcgcgatac cttatgggca gcatgacccc
     6961 ccaggccgtg ctggcgttcg tggccctcat cccgccgacc ttgcccggca caaacatcgt
     7021 gttgggggcc cttccggagg acagacacat cgaccgcctg gccaaacgcc agcgccccgg
     7081 cgagcggctt gacctggcta tgctggccgc gattcgccgc gtttacgggc tgcttgccaa
     7141 tacggtgcgg tatctgcagg gcggcgggtc gtggcgggag gattggggac agctttcggg
     7201 gacggccgtg ccgccccagg gtgccgagcc ccagagcaac gcgggcccac gaccccatat
     7261 cggggacacg ttatttaccc tgtttcgggc ccccgagttg ctggccccca acggcgacct
     7321 gtacaacgtg tttgcctggg ccttggacgt cttggccaaa cgcctccgtc ccatgcacgt
     7381 ctttatcctg gattacgacc aatcgcccgc cggctgccgg gacgccctgc tgcaacttac
     7441 ctccgggatg gtccagaccc acgtcaccac ccccggctcc ataccgacga tctgcgacct
     7501 ggcgcgcacg tttgcccggg agatggggga ggctaactga taaggcgcgc ctctagaggg
     7561 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagtccata gtcgacggat
     7621 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     7681 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     7741 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     7801 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     7861 tatcttagag tcggctcgac atactagtta aaagttttgt tactttatag aagaaatttt
     7921 gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
     7981 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
     8041 tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac
     8101 gtcacaatat gattatcttt ctagggttaa aagggcgaat tcgcggccgc taaattcaat
     8161 tcccgagcgg ccgcgtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     8221 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     8281 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     8341 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     8401 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     8461 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     8521 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gaggcgaaat
     8581 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     8641 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     8701 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     8761 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     8821 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     8881 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     8941 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc
     9001 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     9061 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     9121 tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagtcatg accaaaatcc
     9181 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     9241 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     9301 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     9361 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     9421 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     9481 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     9541 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     9601 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     9661 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     9721 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     9781 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     9841 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     9901 cgttatcccc tgattctgtg gataaccgtg cggccgccaa tactagtcct gcaggtttaa
     9961 acgaattcgc ccttttaa
//
