LOCUS       Exported                8254 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector L071 RRI-Green, complete sequence.
ACCESSION   EU424172
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8254)
  AUTHORS   Micklem DR, Lorens JB.
  TITLE     Retroviral vectors for expression of shRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8254)
  AUTHORS   Micklem DR, Lorens JB.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-JAN-2008) Biomedicine, University of Bergen, Jonas 
            Lies vei 91, Bergen 5009, Norway
REFERENCE   3  (bases 1 to 8254)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8254
                     /organism="Cloning vector L071 RRI-Green"
                     /mol_type="other DNA"
                     /db_xref="taxon:517185"
     repeat_region   2..747
                     /label=CRU5 chimeric CMV promoter, MoMLV and MSCV 5 LTR
                     /note="CRU5 chimeric CMV promoter, MoMLV and MSCV 5 LTR"
     regulatory      2..571
                     /regulatory_class="enhancer"
                     /note="CMV immediate-early enhancer region"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    602..669
                     /label=R region
                     /note="R region"
     misc_feature    748..1665
                     /note="psi+; chimeric MoMLV/MSCV extended packaging signal"
     CDS             1240..1656
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     misc_feature    complement(2063..2270)
                     /note="BGHpA signal; bovine growth hormone polyadenylation 
                     signal"
     polyA_signal    2063..2270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     mRNA            complement(join(2157..3742,4666..4698))
                     /product="GFP2APuro fusion protein"
                     /label=GFP2APuro fusion protein mRNA
     polyA_site      complement(2157)
     CDS             complement(2290..3708)
                     /codon_start=1
                     /product="GFP2APuro fusion protein"
                     /label=GFP2APuro fusion protein
                     /note="fusion between EGFP, FMDV 2A-related self-cleaving 
                     peptide and puromycin acetyl transferase; selectable marker
                     conferring green fluorescence and resistance to puromycin"
                     /protein_id="ACB70965.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKEFGGGSGGGQLLNFDLLKLAGDVESNPGPTTSMTEYKPTVRL
                     ATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADD
                     GAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATV
                     GVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEG
                     PRDLVHDPQARCLI"
     misc_feature    complement(2901..2985)
                     /label=FMDV 2A self-cleaving linker region
                     /note="FMDV 2A self-cleaving linker region"
     CDS             complement(2992..3708)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      complement(3734..4901)
                     /regulatory_class="promoter"
                     /note="EF1-alpha (elongation factor 1 alpha) promoter and 
                     first intron"
     promoter        complement(3734..4896)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          complement(3743..4665)
                     /note="EF1-alpha intron"
     misc_difference complement(4543..4589)
                     /note="not identical to canonical EF1-alpha intron 
                     sequence"
     regulatory      complement(4722..4728)
                     /regulatory_class="TATA_box"
     repeat_region   4962..5869
                     /note="SIN self-inactivating LTR with U6 expression 
                     cassette"
     regulatory      5089..5354
                     /regulatory_class="promoter"
                     /note="modified human U6 promoter"
     protein_bind    5307..5325
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     misc_binding    5307..5324
                     /label=Tet repressor binding site
                     /bound_moiety="Tet repressor"
                     /note="TetO binding site"
     misc_feature    5355..5745
                     /note="stuffer fragment; lost during cloning of shRNAs 
                     between unique BglII and AarI sites"
     misc_feature    5355
                     /label=U6 promoter-dependent transcription initiation
                     /note="U6 promoter-dependent transcription initiation"
     misc_feature    5802..5869
                     /label=R region
                     /note="R region"
     protein_bind    6055..6071
                     /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(6079..6109)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6124..6145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6418..7130)
                     /direction=LEFT
                     /label=pMB1 ori (pUC)
                     /note="pMB1 ori (pUC)"
     rep_origin      complement(6433..7021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7195..8055)
                     /codon_start=1
                     /gene="bla"
                     /product="bla"
                     /label=bla
                     /note="beta-lactamase; ampicillin resistance"
                     /protein_id="ACB70964.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(7195..8055)
                     /gene="bla"
                     /label=bla
     promoter        complement(8056..8160)
                     /gene="bla"
                     /label=AmpR promoter