LOCUS       Exported                5439 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression shuttle vector pGAPDH.Rep68(Y156F), complete sequence.
ACCESSION   GQ380657
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5439)
  AUTHORS   van Nierop GP, de Vries AA, Holkers M, Vrijsen KR, Goncalves MA.
  TITLE     Stimulation of homology-directed gene targeting at an endogenous 
            human locus by a nicking endonuclease
  JOURNAL   Nucleic Acids Res. 37 (17), 5725-5736 (2009)
  PUBMED    19651880
REFERENCE   2  (bases 1 to 5439)
  AUTHORS   van Nierop GP, de Vries AA, Holkers M, Vrijsen KR, Goncalves MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-JUL-2009) Molecular Cell Biology, Leiden University 
            Medical Center, Einthovenweg 20, Leiden, Zuid Holland 2333 ZC, The 
            Netherlands
REFERENCE   3  (bases 1 to 5439)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5439
                     /organism="Expression shuttle vector pGAPDH.Rep68(Y156F)"
                     /mol_type="other DNA"
                     /db_xref="taxon:672172"
     CDS             8..1618
                     /codon_start=1
                     /gene="rep68"
                     /product="Rep68"
                     /label=rep68
                     /note="endonuclease/replicase"
                     /protein_id="ACV74421.1"
                     /translation="MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDL
                     NLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMV
                     LGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNFLLPKTQP
                     ELQWAWTNMEQYLCACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSA
                     RYGELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAP
                     DYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGK
                     TNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKV
                     RVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDF
                     GKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPST
                     SDAEASINYADRLARGHSL"
     gene            8..1618
                     /gene="rep68"
                     /label=rep68
     polyA_signal    1688..1736
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(2223..2239)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2713..2817
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2818..3678
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="ACV74422.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGARERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            2818..3678
                     /gene="bla"
                     /label=bla
     rep_origin      3849..4437
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4725..4746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4761..4791
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4799..4815
                     /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)."
     primer_bind     4823..4839
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"