LOCUS       Exported               14567 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Lentiviral shRNA vector (pGIPz based) for inducible knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    pInducer10-mir-RUP-PheS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14567)
  AUTHORS   Meerbrey KL, Hu G, Kessler JD, Roarty K, Li MZ, Fang JE, 
            Herschkowitz JI, Burrows AE, Ciccia A, Sun T, Schmitt EM, Bernardi 
            RJ, Fu X, Bland CS, Cooper TA, Schiff R, Rosen JM, Westbrook TF, 
            Elledge SJ
  TITLE     The pINDUCER lentiviral toolkit for inducible RNA interference in 
            vitro and in vivo.
  JOURNAL   Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3665-70. doi: 
            10.1073/pnas.1019736108. Epub 2011 Feb 9.
  PUBMED    21307310
REFERENCE   2  (bases 1 to 14567)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1019736108"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2011-03-1- 1"; volume: "108"; issue: "9"; pages: "3665-70"
FEATURES             Location/Qualifiers
     source          1..14567
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    497..730
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             915..959
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1257..1374
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1439..1813)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      1810..1819
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(1823..1841)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1833..1880)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    1950..2220
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        2253..2291
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2288..2312
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2388..3083
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     ncRNA           3134..3227
                     /label=5 miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     3227..3243
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3228..3244
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3265..3282
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3283..3306
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      3316..3325
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4601..4621
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     ncRNA           4645..4757
                     /label=3 miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        4808..6019
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5856..5875
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    complement(6026..6059)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             6066..6812
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    6813..6846
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    6862..7434
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7029..7046)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7256..7275
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             7437..8039
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGSRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7936..7956
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8061..8649
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8114..8134)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8532..8543)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8856..9089
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9190..9207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9196..9420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9466..9894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9553..9572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9763..9784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9903..9923)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9908..10237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10088..10223
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10150..10169
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    11060..11181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11230..11246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11230..11246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11243..11265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11254..11270
                     /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(11278..11308)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11323..11344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11461..11478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11632..12220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11712..11731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12391..13251)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRMDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     13014..13033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13252..13356)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    13404..13538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     LTR             13706..14339
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"