LOCUS       Exported                9583 bp ds-DNA     circular SYN 03-JAN-2022
DEFINITION  gRNA expression vector (with mKate2).
ACCESSION   .
VERSION     .
KEYWORDS    pgRNA-CKB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9583)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen 
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, 
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
  PUBMED    26971820
REFERENCE   2  (bases 1 to 9583)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date: 
            "2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
FEATURES             Location/Qualifiers
     source          1..9583
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    309..426
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    679..712
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     716..736
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        1053..1356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1358..1635
                     /label=chicken beta-actin promoter
     intron          1636..2652
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2576..2598
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2660..2679
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2717..2737
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2741..2761
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2765..2785
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2801..3496
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             3497..3550
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(3994..4013)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4248..4281
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4337..4925
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4390..4410)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4808..4819)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4928..4944)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4929..4945)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5454..5634
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5696..6284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5776..5795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6455..7315)
                     /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"
     primer_bind     7078..7097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7316..7420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7495..7514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7686..8065
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8067..8265
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8220..8240
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8283..8463
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    8510..8635
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9132..9365
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             join(9550..9583,1..11)
                     /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"