LOCUS       Exported                6726 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Mammalian expression vector pCK25, complete sequence.
ACCESSION   HQ644134
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6726
                     /organism="Mammalian expression vector pCK25"
                     /mol_type="other DNA"
                     /note="designed for constitutive expression of the 
                     transrepressor KRAB-mHucR (mUTS); codon optimized for 
                     expression in Homo sapiens"
                     /db_xref="taxon:943930"
     regulatory      470..1653
                     /regulatory_class="promoter"
                     /note="human elongation factor 1 alpha promoter"
     promoter        475..1653
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          706..1644
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     promoter        1670..1688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            1775..2689
                     /gene="mUTS"
                     /label=mUTS
                     /note="mammalian urate transsilencer; KRAB-HucR"
     CDS             1805..1999
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             2712..2753
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2763..2780
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2809..3033
                     /regulatory_class="polyA_signal_sequence"
     polyA_signal    2809..3033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3079..3507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     regulatory      3512..3856
                     /regulatory_class="promoter"
                     /note="simian virus 40 promoter"
     promoter        3521..3851
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3702..3837
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3899..3946
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     gene            3965..4363
                     /gene="blasticidin resistance protein"
                     /label=blasticidin resistance protein
     CDS             3965..4363
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      4521..4651
                     /regulatory_class="polyA_signal_sequence"
     polyA_signal    4521..4642
                     /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    4715..4731
                     /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(4739..4769)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4784..4805
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /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"
     gene            complement(5852..6712)
                     /gene="AmpR"
                     /label=AmpR
                     /note="ampicillin resistance protein"
     CDS             complement(5852..6712)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6713..91)
                     /gene="bla"
                     /label=AmpR promoter