LOCUS       Exported                8893 bp ds-DNA     circular SYN 20-DEC-2021
DEFINITION  CRISPRi/a V2 library parental plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    pCRISPRia-v2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8893)
  AUTHORS   Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, 
            Fields AP, Park CY, Corn JE, Kampmann M, Weissman JS
  TITLE     Compact and highly active next-generation libraries for 
            CRISPR-mediated gene repression and activation.
  JOURNAL   Elife. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760.
  PUBMED    27661255
REFERENCE   2  (bases 1 to 8893)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 20, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8893
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    293..526
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             711..755
                     /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    1053..1170
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    1431..1464
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     1468..1488
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1741..1952
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1953..2891
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2848..2868
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2912..3508
                     /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(2912..2931)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3408..3428
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3518..3571
                     /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             3584..4282
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    4302..4335
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4391..4979
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4444..4464)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4862..4873)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4982..4998)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4983..4999)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5508..5688
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5750..6338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5830..5849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6509..7369)
                     /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     7132..7151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7370..7474)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7549..7568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7740..8119
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8121..8319
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8274..8294
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8337..8517
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8564..8689
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
       61 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
      121 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      181 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
      241 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
      301 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
      361 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
      421 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
      481 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
      541 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
      601 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
      661 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
      721 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
      781 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
      841 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
      901 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
      961 gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa
     1021 ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     1081 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     1141 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     1201 gtttggttag taccgggccc gccctgcagg tctagagatc cgacgcgcca tctctaggcc
     1261 cgcgccggcc ccctcgcacg gacttgtggg agaagctcgg ctactcccct gccccggtta
     1321 atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa agacagataa
     1381 tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct ataacttcgt
     1441 atagcataca ttatacgaag ttataaacag cacaaaagga aactcaccct aactgtaaag
     1501 taattgtgtg ttttgagact ataagtatcc cttggagaac caccttgttg gaccaggatg
     1561 ggcaccaccc gtttaagagc taagctggaa acagcatagc aagtttaaat aaggctagtc
     1621 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctcgagt actaggatcc
     1681 attaggcggc cgccctgcag ggtggataac cgtattaccg ccatgcatgt gcccgtcagt
     1741 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     1801 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     1861 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     1921 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     1981 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     2041 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     2101 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     2161 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     2221 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     2281 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     2341 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     2401 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     2461 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     2521 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     2581 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     2641 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg cgcttttgga
     2701 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     2761 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     2821 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     2881 ttccatttca ggtgtcgtga gctagcccac catgaccgag tacaagccca cggtgcgcct
     2941 cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta
     3001 ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca
     3061 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3121 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3181 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3241 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     3301 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     3361 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     3421 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     3481 ctggtgcatg acccgcaagc ccggtgccgg atcgggagag ggcagaggaa gtctgctaac
     3541 atgcggtgac gtcgaggaga atcctggccc accggtcgcc accagcgagc tgattaagga
     3601 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
     3661 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
     3721 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
     3781 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
     3841 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     3901 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     3961 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     4021 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     4081 gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct
     4141 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     4201 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     4261 actggggcac aagcttaatt aagaattcgt cgagggacct aataacttcg tatagcatac
     4321 attatacgaa gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa
     4381 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     4441 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     4501 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     4561 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     4621 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     4681 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     4741 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     4801 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     4861 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     4921 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     4981 tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata
     5041 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt
     5101 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta
     5161 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     5221 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca
     5281 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag
     5341 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg
     5401 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc
     5461 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt
     5521 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     5581 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     5641 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa
     5701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7501 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     7561 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     7621 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     7681 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     7741 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     7801 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     7861 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     7921 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     7981 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     8041 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     8101 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     8161 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     8221 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     8281 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
     8341 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8401 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     8461 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     8521 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     8581 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     8641 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     8701 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     8761 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     8821 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     8881 gacaggatca gaa
//
