LOCUS       Exported                8491 bp ds-DNA    circular SYN 22-2-2014
DEFINITION  HuSH-29 lentiviral vector with chloramphenicol and blasticidin 
            resistance markers and a TurboRFP gene, for expressing an shRNA from
            the U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pRFP-CB-shLenti
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8491)
  AUTHORS   OriGene
  TITLE     Direct Submission
COMMENT     The shRNA cassette consists of a 29-bp gene-specific sequence, 
            followed by a 7-bp loop, followed by the reverse complement of the 
            29-bp sequence, followed by a TTTTTT terminator.
FEATURES             Location/Qualifiers
     source          1..8491
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     enhancer        237..616
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             834..1014
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2443..2560
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2707..2947
                     /label="U6 Promoter"
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    2951..3008
                     /note="shRNA site"
                     /note="shRNA insertion site"
     rep_origin      3133..3268
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             3299..3697
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /note="BSD"
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     enhancer        4104..4483
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4484..4687
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        4713..4731
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4839..5534
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /note="TurboRFP"
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     protein_bind    5586..5619
                     /bound_moiety="Cre recombinase"
                     /note="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    5675..6263
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             6792..6972
                     /note="3 LTR (truncated)"
                     /note="truncated 3 long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7034..7622)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7699..8358)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(8359..8463)
                     /gene="bla"
                     /note="AmpR promoter"