LOCUS       Exported                8903 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Lentiviral CRISPR guide vector expressing a eGFP-NT2 sgRNA with cs1 
            incorporated in the loop of the sgRNA constant region. BsmBI sites 
            were removed to allow for programmed dual sgRNA library cloning..
ACCESSION   .
VERSION     .
KEYWORDS    pJR85
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8903)
  TITLE     Direct capture of CRISPR guides enables scalable, multiplexed, and 
            multi-omic Perturb-seq
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8903
                     /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        3142..3353
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3354..4292
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4249..4269
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4313..4909
                     /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(4313..4332)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4809..4829
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4919..4972
                     /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             4985..5683
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    5703..5736
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    5792..6380
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5845..5865)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6263..6274)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6383..6399)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6384..6400)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6909..7089
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7151..7739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7231..7250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7910..8770)
                     /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     8533..8552
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8771..8875)
                     /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 aacttggccg ctttaaggcc ggtcctagca aggccaagtg gcaccgagtc
     3061 ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc gcgtggataa ccgtattacc
     3121 gccatgcatg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     3181 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     3241 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     3301 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
     3361 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
     3421 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
     3481 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
     3541 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
     3601 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
     3661 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     3721 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     3781 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     3841 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     3901 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     3961 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     4021 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     4081 attagttctc gcgcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     4141 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     4201 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     4261 cagtggttca aagttttttt cttccatttc aggtgtcgtg agctagccca ccatgaccga
     4321 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct
     4381 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat
     4441 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4501 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4561 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4621 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4681 gttcctggcc accgtcggcg tgtcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4741 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     4801 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4861 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga
     4921 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtcgc
     4981 caccagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     5041 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     5101 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     5161 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     5221 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     5281 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     5341 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     5401 ggaggccttc accgagaccc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     5461 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     5521 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     5581 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     5641 atactgcgac ctccctagca aactggggca caagcttaat taagaattcg tcgagggacc
     5701 taataacttc gtatagcata cattatacga agttatacat gtttaagggt tccggttcca
     5761 ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     5821 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5881 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5941 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     6001 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     6061 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     6121 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     6181 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     6241 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     6301 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     6361 cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg
     6421 gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac
     6481 aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt
     6541 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     6601 ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     6661 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
     6721 ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga
     6781 acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat
     6841 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
     6901 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6961 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     7021 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     7081 tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     7141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     7201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     7261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     7321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     7381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7561 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7801 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7861 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7921 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7981 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     8041 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     8101 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     8161 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     8221 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     8281 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     8341 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     8401 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8461 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8521 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8581 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8641 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8701 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8761 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8821 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8881 ttccccgaaa agtgccacct gac
//
