LOCUS       Exported                7454 bp ds-DNA    circular SYN 22-2-2014
DEFINITION  HuSH-29 retroviral vector with chloramphenicol and puromycin 
            resistance markers and a TurboRFP gene, for expressing an shRNA from
            the U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pRFP-C-RS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  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..7454
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     promoter        90..330
                     /label="U6 Promoter"
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    334..385
                     /note="shRNA site"
                     /note="shRNA insertion site"
     promoter        463..659
                     /note="SV40 promoter"
                     /note="SV40 early promoter"
     rep_origin      510..645
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             673..1272
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     LTR             1351..1944
                     /note="3 LTR"
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     rep_origin      complement(2316..2904)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    3091..3567
                     /note="hGH poly(A) signal"
                     /note="human growth hormone polyadenylation signal"
     CDS             complement(3688..4383)
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /note="TurboRFP"
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     promoter        complement(4427..4445)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4471..4674)
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        4675..5054
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             complement(5266..5925)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     LTR             6100..6569
                     /note="5 LTR"
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6634..6833
                     /note="MMLV Psi"
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             7026..7442
                     /note="gag (truncated)"
                     /note="truncated MMLV gag gene lacking the start codon"