LOCUS       40924_8011        8689 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCAG-EBFP2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8689)
  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  JOURNAL   Unpublished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 8689)
  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 8689)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8689)
  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            "Unpublished"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..8689
                     /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"
     enhancer        4313..4616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4619..4894
                     /label=chicken beta-actin promoter
     intron          4900..5908
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     protein_bind    5966..5990
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5998..6714
                     /codon_start=1
                     /label=EBFP2
                     /note="enhanced blue variant of GFP (Ai et al., 2007)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     protein_bind    complement(6722..6746)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6901..6956
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7317..7333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7341..7357
                     /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(7365..7395)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7410..7431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    7604..8440
                     /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(8470..8488)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8509..8525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8533..8549)
                     /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(8557..8587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8602..8623)
                     /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 cgagttaatt aatagttatt
     4261 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     4321 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     4381 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     4441 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     4501 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     4561 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatgggtc
     4621 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     4681 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg ggggggggcg
     4741 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
     4801 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     4861 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gagtcgctgc gttgccttcg
     4921 ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     4981 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
     5041 taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc
     5101 cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
     5161 cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     5221 gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     5281 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     5341 gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt gctgagcacg
     5401 gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg
     5461 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     5521 cgggggaggg gcgcggcggc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     5581 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     5641 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt
     5701 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     5761 cccttctcca tctccagcct cggggctgcc gcagggggac ggctgccttc gggggggacg
     5821 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     5881 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc
     5941 tcatcatttt ggcaaagaat tcgacccaag tttgtacaaa aaagcaggct cgccaccatg
     6001 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     6061 gacgtaaacg gccacaagtt cagcgtgagg ggcgagggcg agggcgatgc caccaacggc
     6121 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     6181 gtgaccaccc tgagccacgg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
     6241 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     6301 aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctagtg
     6361 aaccgcatcg agctgaaggg cgtcgacttc aaggaggacg gcaacatcct ggggcacaag
     6421 ctggagtaca acttcaacag ccacaacatc tatatcatgg ccgtcaagca gaagaacggc
     6481 atcaaggtga acttcaagat ccgccacaac gtggaggacg gcagcgtgca gctcgccgac
     6541 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga cagccactac
     6601 ctgagcaccc agtccgtgct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     6661 ctggagttcc gcaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatac
     6721 cacccagctt tcttgtacaa agtggtacgc gtgaattcac tcctcaggtg caggctgcct
     6781 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     6841 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     6901 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     6961 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     7021 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     7081 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     7141 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     7201 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     7261 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     7321 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     7381 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     7441 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcga cagtactaag
     7501 cttggtctaa tacccaatct ctcgtcttat ccagatgttt tatacgcctt aattaatagg
     7561 gataacaggg taatctcgag gtttaaacgt cgacgcggcc gctactaggg acaggattgg
     7621 tgacagaaaa gccccatcct taggcctcct ccttcctagt ctcctgatat tgggtctaac
     7681 ccccacctcc tgttaggcag attccttatc tggtgacaca cccccatttc ctggagccat
     7741 ctctctcctt gccagaacct ctaaggtttg cttacgatgg agccagagag gatcctggga
     7801 gggagagctt ggcagggggt gggagggaag ggggggatgc gtgacctgcc cggttctcag
     7861 tggccaccct gcgctaccct ctcccagaac ctgagctgct ctgacgcggc tgtctggtgc
     7921 gtttcactga tcctggtgct gcagcttcct tacacttccc aagaggagaa gcagtttgga
     7981 aaaacaaaat cagaataagt tggtcctgag ttctaacttt ggctcttcac ctttctagtc
     8041 cccaatttat attgttcctc cgtgcgtcag ttttacctgt gagataaggc cagtagccag
     8101 ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg tgtggaaaac tccctttgtg
     8161 agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc tctactccct ttctctttct
     8221 ccatccttct ttccttaaag agtccccagt gctatctggg acatattcct ccgcccagag
     8281 cagggtcccg cttccctaag gccctgctct gggcttctgg gtttgagtcc ttggcaagcc
     8341 caggagaggc gctcaggctt ccctgtcccc cttcctcgtc caccatctca tgcccctggc
     8401 tctcctgccc cttccctaca ggggttcctg gctctgctct ccaccgcggt ggagctccag
     8461 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     8521 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     8581 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     8641 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatc
//
