LOCUS       Exported               11810 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector GAPTrapT2AIresMuro, complete sequence.
ACCESSION   KX783040
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11810)
  AUTHORS   Kao T, Labonne T, Niclis JC, Chaurasia R, Lokmic Z, Qian E, Bruveris
            FF, Howden SE, Motazedian A, Schiesser JV, Costa M, Sourris K, Ng E,
            Anderson D, Giudice A, Farlie P, Cheung M, Lamande SR, Penington AJ,
            Parish CL, Thomson LH, Rafii A, Elliott DA, Elefanty AG, Stanley EG.
  TITLE     GAPTrap: A Simple Expression System for Pluripotent Stem Cells and 
            Their Derivatives
  JOURNAL   Stem Cell Reports (2016) In press
  PUBMED    27594589
REFERENCE   2  (bases 1 to 11810)
  AUTHORS   Stanley E.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-AUG-2016) Cell Biology, Murdoch Childrens Research 
            Institute, Royal Childrens Hospital, Flemington Road, Melbourne, 
            Victoria 3052, Australia
REFERENCE   3  (bases 1 to 11810)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..11810
                     /organism="Cloning vector GAPTrapT2AIresMuro"
                     /mol_type="other DNA"
                     /db_xref="taxon:1899386"
     gene            24..989
                     /gene="ampR"
                     /label=ampR
     regulatory      24..128
                     /regulatory_class="promoter"
                     /gene="ampR"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             129..989
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /protein_id="AOO95912.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1160..11748
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="CAP"
     regulatory      2072..2102
                     /regulatory_class="promoter"
                     /note="lac promoter"
     promoter        2072..2102
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_feature    2110..2126
                     /label=lac operator
                     /note="lac operator"
     protein_bind    2110..2126
                     /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     2134..2150
                     /label=M13 rev
                     /note="M13 rev"
     misc_feature    2146..2200
                     /label=lacZ-alpha
                     /note="lacZ-alpha"
     regulatory      2171..2189
                     /regulatory_class="promoter"
                     /note="T3 promoter"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     gene            2216..5559
                     /gene="GAPDH"
                     /label=GAPDH
                     /note="glyceraldehyde-3-phosphate dehydrogenase"
     misc_feature    2219..5556
                     /gene="GAPDH"
                     /label=left arm
                     /note="left arm"
     misc_feature    5560..5658
                     /label=T2A
                     /note="T2A"
     CDS             5581..5634
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     misc_feature    5657..6243
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     regulatory      5684..6246
                     /regulatory_class="attenuator"
                     /note="IRES"
     CDS             6253..6861
                     /codon_start=1
                     /product="Muro"
                     /label=Muro
                     /note="codes for puromycin resistance"
                     /protein_id="AOO95913.1"
                     /translation="MARLTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRH
                     IERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGS
                     RLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    6897..11132
                     /label=right arm
                     /note="right arm"
     misc_feature    11152..11810
                     /label=pBluescript II KS(-)
                     /note="pBluescript II KS(-)"
     promoter        complement(11167..11185)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11195..11211)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(11352..11807)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"