LOCUS       Exported                8877 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Parental vector for the CRISPRi/a libraries. Expresses an sgRNA from
            the U6 promoter and a puromycin resistance cassette and BFP from the
            EF1Alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pU6-sgRNA EF1Alpha-puro-T2A-BFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8877)
  AUTHORS   Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead 
            EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M,
            Weissman JS
  TITLE     Genome-Scale CRISPR-Mediated Control of Gene Repression and 
            Activation.
  JOURNAL   Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029. 
            Epub 2014 Oct 9.
  PUBMED    25307932
REFERENCE   2  (bases 1 to 8877)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8877
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2814..2847
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     2851..2871
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        3116..3327
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3328..4266
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4223..4243
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4287..4883
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4287..4306)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4783..4803
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4893..4946
                     /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"
     CDS             4959..5657
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    5677..5710
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    5766..6354
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5819..5839)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6237..6248)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6357..6373)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6358..6374)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6883..7063
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7125..7713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7205..7224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7884..8744)
                     /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     8507..8526
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8745..8849)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag atccgacgcg ccatctctag
     2641 gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc cctgccccgg
     2701 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
     2761 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataactt
     2821 cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac cctaactgta
     2881 aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg ttggaccagg
     2941 atgggcacca cccgtttaag agctaagctg gaaacagcat agcaagttta aataaggcta
     3001 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg agtactagga
     3061 tccattaggc ggccgcgtgg ataaccgtat taccgccatg catgtgcccg tcagtgggca
     3121 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     3181 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     3241 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     3301 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
     3361 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
     3421 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
     3481 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
     3541 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     3601 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     3661 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
     3721 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     3781 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     3841 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     3901 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     3961 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     4021 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgcgctt ttggagtacg
     4081 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     4141 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     4201 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     4261 tttcaggtgt cgtgagctag cccaccatga ccgagtacaa gcccacggtg cgcctcgcca
     4321 cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     4381 ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     4441 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     4501 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     4561 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     4621 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     4681 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     4741 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     4801 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     4861 gcatgacccg caagcccggt gccggatcgg gagagggcag aggaagtctg ctaacatgcg
     4921 gtgacgtcga ggagaatcct ggcccaccgg tcgccaccag cgagctgatt aaggagaaca
     4981 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     5041 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     5101 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     5161 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     5221 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     5281 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     5341 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     5401 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     5461 atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga
     5521 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     5581 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     5641 ggcacaagct taattaagaa ttcgtcgagg gacctaataa cttcgtatag catacattat
     5701 acgaagttat acatgtttaa gggttccggt tccactaggt acaattcgat atcaagctta
     5761 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5821 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5881 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5941 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     6001 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     6061 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     6121 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     6181 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     6241 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     6301 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     6361 accgtcgacc tcgatcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca
     6421 gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca
     6481 gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     6541 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc
     6601 cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca
     6661 gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag
     6721 caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc
     6781 ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta
     6841 gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac
     6901 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     6961 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     7021 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc
     7081 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     7141 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     7201 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7261 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     7321 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7381 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7441 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7501 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7561 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7621 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7681 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7741 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7801 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7861 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7921 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7981 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     8041 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     8101 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     8161 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     8221 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8281 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8341 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8401 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8461 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8521 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8581 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8641 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8701 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8761 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8821 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
