LOCUS       40924_10791        6726 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Mammalian expression vector pCK25, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Kemmer C, Gitzinger M, Daoud-El Baba M, Djonov V, Stelling J, 
            Fussenegger M.
  TITLE     Self-sufficient control of urate homeostasis in mice by a synthetic 
            circuit
  JOURNAL   Nat. Biotechnol. 28 (4), 355-360 (2010)
  PUBMED    20351688
REFERENCE   2  (bases 1 to 6726)
  AUTHORS   Kemmer C, Gitzinger M, Daoud-El Baba M, Djonov V, Stelling J, 
            Fussenegger M.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-NOV-2010) Department of Biosystems Science and 
            Engineering, D-BSSE, ETH Zurich, Mattenstrasse 20, Basel 4058, 
            Switzerland
REFERENCE   3  (bases 1 to 6726)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6726)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol."; date: "2010"; volume: "28"; issue: "4"; pages: 
            "355-360"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-NOV-2010) Department of Biosystems Science and Engineering, 
            D-BSSE, ETH Zurich, Mattenstrasse 20, Basel 4058, Switzerland"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        475..1653
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     promoter        1670..1688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1805..1999
                     /codon_start=1
                     /label=KRAB
                     /note="Kruppel-associated box (KRAB) transcriptional
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             2712..2753
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     CDS             2763..2780
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    2809..3033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3079..3507
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3521..3851
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        3899..3946
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             3965..4360
                     /codon_start=1
                     /label=BSD
                     /note="blasticidin S deaminase"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4521..4654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4691..4707)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4715..4731)
                     /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(4739..4769)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4784..4805)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5093..5681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5855..6712)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(join(6713..6726,1..91))
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
      421 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc
      481 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      541 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      601 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      661 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
      721 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
      781 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
      841 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
      901 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
      961 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     1021 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     1081 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     1141 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     1201 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     1261 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     1321 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     1381 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     1441 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     1501 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     1561 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     1621 caaagttttt ttcttccatt tcaggtgtcg tgaggaatta gcttggtact aatacgactc
     1681 actataggga gacccaagct ggctaggtaa gcttggtacc gagctcggat ccactagtcc
     1741 agtgtggtgg aattgccctt gtacgaattc caccatggat gctaagtcac taactgcctg
     1801 gtcccggaca ctggtgacct tcaaggatgt atttgtggac ttcaccaggg aggagtggaa
     1861 gctgctggac actgctcagc agatcgtgta cagaaatgtg atgctggaga actataagaa
     1921 cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg
     1981 agaagagccc tggctggtgg agagagaaat tcaccaagag acccatcctg attcagagac
     2041 tgcatttgaa atcaaatcat cagtttccag caggagcatt tttaaagata agcaatcctg
     2101 tgacattaaa atggaaggaa tggcaaggaa tgatctctgg gcgcgctcag cccgcatgga
     2161 caacgacacg gcagcccttc tggagcggat tcggagcgac tgggcgaggc tcaatcacgg
     2221 tcagggacca gactcggacg gtctgacccc cagcgccggg ccgatgctga ccctgctgct
     2281 gctcgagcgg ctgcacgcgg ctctggggcg tgaaatcgag cggacctacg ccgcttcggg
     2341 gctgaacgcg gcgggctggg acctgctgct cacgctttac cgctcggcgc cgcccgaggg
     2401 cctgcggccc accgaactca gcgccctggc cgccatttcc gggccttcga cgagcaaccg
     2461 gatcgtgcgg ctgctcgaaa agggcctgat cgagcgccgc gaggacgaac gtgaccgccg
     2521 ctcggcgagt attcgcctga cgccgcaggg ccgcgccctg gtcacgcacc tgctgcccgc
     2581 acatctggcg accacgcagc gggtgcttgc tccgctgtcg gcgcaggagc agcgcaccct
     2641 ggaggagctg gcggggcgaa tgctggcggg gctggagcag ggggtataat ctagagggcc
     2701 cgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg
     2761 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     2821 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2881 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2941 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3001 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3061 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3121 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3181 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc
     3241 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3301 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3361 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3421 tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc
     3481 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3541 aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3601 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3661 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3721 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3781 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3841 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt
     3901 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     3961 aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     4021 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     4081 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     4141 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     4201 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgtcgacagg tgcttctcga
     4261 tctgcatcct gggatcaaag cgatagtgaa ggacagtgat ggacagccga cggcagttgg
     4321 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc gtggccgagg
     4381 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4441 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4501 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4561 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4621 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4681 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4741 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4801 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4861 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4921 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4981 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5041 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5101 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5161 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5221 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5281 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5341 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5401 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5461 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5521 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5581 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5761 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5821 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5881 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5941 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6001 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6061 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6121 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6181 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6241 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6301 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6361 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6421 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6481 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6541 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6601 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6661 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6721 tttttc
//
