LOCUS       40924_44008        7352 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTRE-EBFP2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7352)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Modular construction of mammalian gene circuits using TALE 
            transcriptional repressors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7352)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-SEP-2014) Bioinformatics Division/Center for Synthetic
REFERENCE   3  (bases 1 to 7352)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7352)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-SEP-2014) Bioinformatics Division/Center for Synthetic "
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(222..810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(984..1841)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1842..1946)
                     /label=AmpR promoter
     rep_origin      complement(1972..2427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2569..2585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2595..2613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2628..3431
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    3510..3535
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    4207..4227
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     promoter        4242..4556
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4629..4653
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4661..5377
                     /codon_start=1
                     /label=EBFP2
                     /note="enhanced blue variant of GFP (Ai et al., 2007)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     protein_bind    complement(5385..5409)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    5564..5619
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5980..5996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6004..6020
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6028..6058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6073..6094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    6267..7103
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(7133..7151)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7172..7188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7196..7212)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(7220..7250)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7265..7286)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
       61 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      121 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      661 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      901 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      961 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1021 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1081 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1141 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1201 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1261 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1321 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1381 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1441 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1501 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1561 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1621 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1681 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1741 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1801 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1861 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     1981 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2041 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2101 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2161 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2221 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2281 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2341 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2401 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     2461 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     2521 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     2581 ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtacctgc tttctctgac
     2641 cagcattctc tcccctgggc ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc
     2701 tctacggctg gcccagatcc ttccctgccg cctccttcag gttccgtctt cctccactcc
     2761 ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct
     2821 tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc
     2881 cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc
     2941 cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg
     3001 tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca
     3061 accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc
     3121 ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct
     3181 cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc
     3241 ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact
     3301 tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat
     3361 attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc
     3421 cacagtgggg caagcttatt accctgttat ccctaaccgg tgcgttttta tgcttgtagt
     3481 attgtataat gtttttaaga tccaagcttc tgacctcttc tcttcctccc acagggcctc
     3541 gagacaattg catcgatggt accagtgaaa gtctgcaatg aattcgagtt ggcgcgcctg
     3601 tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca
     3661 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc
     3721 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg
     3781 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg
     3841 gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct
     3901 ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     3961 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     4021 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     4081 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     4141 tccccgtgag ctcccaggcg cgccaactcg gtaccgtatc gatgtcgacg ttaacgctag
     4201 tgatatcaac tttgtataga aaagttggct ccgaatttct cgagtttact ccctatcagt
     4261 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac
     4321 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
     4381 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga
     4441 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc
     4501 gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     4561 gatttcgagc tcggtacccg gggatcctct agtcagctga cgcgtgctag tggcgcgccg
     4621 aattcgaccc aagtttgtac aaaaaagcag gctcgccacc atggtgagca agggcgagga
     4681 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     4741 gttcagcgtg aggggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt
     4801 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca
     4861 cggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     4921 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaccta
     4981 caagacccgc gccgaggtga agttcgaggg cgacacccta gtgaaccgca tcgagctgaa
     5041 gggcgtcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa
     5101 cagccacaac atctatatca tggccgtcaa gcagaagaac ggcatcaagg tgaacttcaa
     5161 gatccgccac aacgtggagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     5221 ccccatcggc gacggccccg tgctgctgcc cgacagccac tacctgagca cccagtccgt
     5281 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tccgcaccgc
     5341 cgccgggatc actctcggca tggacgagct gtacaagtaa taccacccag ctttcttgta
     5401 caaagtggta cgcgtgaatt cactcctcag gtgcaggctg cctatcagaa ggtggtggct
     5461 ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat
     5521 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     5581 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     5641 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     5701 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     5761 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     5821 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     5881 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg
     5941 agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     6001 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     6061 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     6121 ccagtcggga aacctgtcgt gccagcggat cgacagtact aagcttggtc taatacccaa
     6181 tctctcgtct tatccagatg ttttatacgc cttaattaat agggataaca gggtaatctc
     6241 gaggtttaaa cgtcgacgcg gccgctacta gggacaggat tggtgacaga aaagccccat
     6301 ccttaggcct cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg
     6361 cagattcctt atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa
     6421 cctctaaggt ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg
     6481 ggtgggaggg aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac
     6541 cctctcccag aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt
     6601 gctgcagctt ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata
     6661 agttggtcct gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc
     6721 ctccgtgcgt cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc
     6781 tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag
     6841 gtgttcacca ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta
     6901 aagagtcccc agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct
     6961 aaggccctgc tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg
     7021 cttccctgtc ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct
     7081 acaggggttc ctggctctgc tctccaccgc ggtggagctc cagcttttgt tccctttagt
     7141 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     7201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     7261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     7321 gaaacctgtc gtgccagctg cattaatgaa tc
//
