LOCUS       Exported                8102 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEFBOS-creIRESPuro, complete sequence.
ACCESSION   EU693012
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8102)
  AUTHORS   Davis RP, Ng ES, Costa M, Mossman AK, Sourris K, Elefanty AG, 
            Stanley EG.
  TITLE     Targeting a GFP reporter gene to the MIXL1 locus of human embryonic 
            stem cells identifies human primitive streak-like cells and enables 
            isolation of primitive hematopoietic precursors
  JOURNAL   Blood 111 (4), 1876-1884 (2008)
  PUBMED    18032708
REFERENCE   2  (bases 1 to 8102)
  AUTHORS   Davis RP, Costa M, Grandela C, Holland AM, Hatzistavrou T, Micallef 
            SJ, Li X, Goulburn AL, Azzola L, Elefanty AG, Stanley EG.
  TITLE     A protocol for removal of antibiotic resistance cassettes from human
            embryonic stem cells genetically modified by homologous 
            recombination or transgenesis
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 8102)
  AUTHORS   Davis RP, Stanley EG, Elefanty AG.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAY-2008) MISCL, Monash University, Wellington, 
            Clayton, Victoria 3800, Australia
REFERENCE   4  (bases 1 to 8102)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8102
                     /organism="Expression vector pEFBOS-creIRESPuro"
                     /mol_type="other DNA"
                     /db_xref="taxon:530757"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      239..1743
                     /regulatory_class="promoter"
                     /note="EF1-alpha promoter"
     promoter        complement(325..521)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(333..543)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      339..474
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        562..1743
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          792..1734
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             1763..2815
                     /codon_start=1
                     /gene="CRE"
                     /product="cre recombinase"
                     /label=CRE
                     /protein_id="ACD68265.1"
                     /translation="MPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFS
                     EHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNM
                     LHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQ
                     DIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSL
                     GVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYG
                     AKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETG
                     AMVRLLEDGD"
     gene            1763..2815
                     /gene="CRE"
                     /label=CRE
     CDS             1766..1786
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1787..2815
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    2849..3435
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_structure  2860..3435
                     /label=internal ribosomal entry site (IRES)
                     /note="internal ribosomal entry site (IRES)"
     CDS             3436..4044
                     /codon_start=1
                     /gene="pac"
                     /product="puromycin-N-acetyltransferase"
                     /label=pac
                     /protein_id="ACD68266.1"
                     /translation="MARLTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRH
                     IERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGS
                     RLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     gene            3436..4044
                     /gene="pac"
                     /label=pac
     CDS             3448..4044
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    4125..4349
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     3UTR           4522..5222
                     /label=bovine growth hormone 3 UTR
                     /note="bovine growth hormone 3 UTR"
     misc_feature    5225..8102
                     /label=plasmid backbone from pUC8
                     /note="plasmid backbone from pUC8"
     primer_bind     complement(5231..5247)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5460..5915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6197..6301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6302..7162
                     /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"
     rep_origin      7333..7921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"