LOCUS       40924_47283        8694 bp DNA     circular SYN 13-MAY-2021
DEFINITION  for CRISPRa, lentiviral expression of gRNA scaffold using activator 
            P65 HSF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8694)
  AUTHORS   Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, 
            Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG
  TITLE     Optimized libraries for CRISPR-Cas9 genetic screens with multiple 
            modalities.
  JOURNAL   Nat Commun. 2018 Dec 21;9(1):5416. doi: 10.1038/s41467-018-07901-8.
  PUBMED    30575746
REFERENCE   2  (bases 1 to 8694)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8694)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Commun."; date: "2018-12-21"; pages: "
            10.1038/s41467-018-07901-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             complement(104..337)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(427..1023)
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             complement(1051..1104)
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1120..1491)
                     /codon_start=1
                     /label=HSF1
                     /note="C-terminal activation domain from the human heat
                     shock transcription factor HSF1"
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             complement(1516..2058)
                     /codon_start=1
                     /label=p65
                     /note="C-terminal portion of the p65 subunit of mouse
                     NF-kappa-B"
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             complement(2074..2094)
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     protein_bind    2145..2165
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     promoter        complement(2633..3137)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     misc_feature    complement(3192..3309)
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        complement(3591..3831)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     CDS             complement(4009..4053)
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     misc_feature    complement(4238..4471)
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    complement(4964..5089)
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     LTR             complement(5136..5316)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5317..5543)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     promoter        complement(5571..5589)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5610..5626)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5634..5650)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5658..5688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5703..5724)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6012..6600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6774..7631)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7632..7736)
                     /label=AmpR promoter
     rep_origin      complement(7762..8217)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     8359..8375
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8385..8403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(8424..8559)
                     /direction=LEFT
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    complement(join(8586..8694,1..26))
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt ctttgcaagt
       61 tataaatact gaataataag atgacatgaa ctactatacg tactgctaga gattttccac
      121 actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac
      181 actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag
      241 ccagagagct cccaggctca gatctggtct aaccagagag acccagtaca agcaaaaagc
      301 agatcttgtc ttcgttggga gtgaattagc ccttccagtc cccccttttc ttttaaaaag
      361 tggctaagat ctacagctgc cttgtaagtc attggtctta aagtcgactt aacgcgttca
      421 gctagcggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac
      481 gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggaggt
      541 ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc
      601 gctgcccaga cccttgccct ggtggtcggg cgaaacgccg acggtggcca ggaaccacgc
      661 gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg
      721 ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac
      781 gctctccggc gtggtccaga ccgccacggc ggcgccgtcg tccgcgaccc acaccttgcc
      841 gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg
      901 gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt
      961 gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt
     1021 catggtaagc tccggtggat ctgtggtacc tgggccagga ttctcctcga cgtcaccgca
     1081 tgttagcaga cttcctctgc cctctccact gccgatatcg gagacagtgg ggtccttggc
     1141 tttgggaggc tccgagcctg tcagcaggga gatggtgggg tcctcggcga agccgtcccc
     1201 ttcggagaag taggagccct ctcccagctc aaacagcacc ggcaggtcgt tgctcccggt
     1261 gtccacggag ccggggtcca gcaggaacag cggctgcgct gtgtagtgca ccagctgctt
     1321 ccctgaatcc gggctgctgt tctctgcctc gggaggcctg gggggctcct ggggagacag
     1381 gagctcttgg atactggcca ggctgctgtc aaggtcaggc aggctcatgt cgggcacggt
     1441 caccgagggg ctgaacaggt ccagcagggc actggtgtcc acgctgaagc cgcttccacc
     1501 tcctccctgc ccactagagg aaatctgtga cagcagggca ctaaagtcca tatcagcgat
     1561 gcttgagaag tcttcatctc cggacagccc attaggcagg ccgctggttc ccaggggagt
     1621 tggagcgggg tcggggggcc gctggctgcc ggtcaccagc cgggtaatgg cttcggggta
     1681 ctccatcagc attggttcgg ctgtactatg agacatggac acgccctgat tcagcagctg
     1741 ctgaaactca gagttgtcca cggaggccag atctgtgaac actccgggat cggtgctgtt
     1801 ccccagcaga gctcccaggt cctcatcagc gtcgaactgc aggtgcagca gagcttcact
     1861 cagagtcccc tcgccggcct gtgtagactt gggcactgga gcgctcagtg actggggtgg
     1921 tcctggggtc agcacagggg ctggagcagg tggctgggcc agaggcacca tagcactaga
     1981 gggcaccata gtctgggcca gcactggagc ggagctaggg gccagagcca gggcctggtt
     2041 gctgatctgc cctgaagggg taccagcggc cgccaccttc ctctttttct taggtccgct
     2101 acctcctcct ccgcttcctc ctcctccgct tccacctcct ccgcccactt tgtacaagaa
     2161 agttgaagaa cggcccagcg gcacaaggtt gacgacaaga tcttcgacct gcgaggtcgc
     2221 gacgagggac ttggtcaaat cgtacaacga tttgcgcgag gcctcggtgc tattcgcaac
     2281 gattgtcacg tcgtgcgacc atacctgagt gtagcgcact ttcggaagtc cggaatcaac
     2341 gacgtccgcc tgatccagtt ttaggttgac tcgatacgcg gtcttggctc cgttttgacg
     2401 gagcgaagcc gtgaggcgca gccgacccac cagaggcccg accttctctt cgaagatctg
     2461 acggtctgcg gtggactgga tctcagtcag agtgcgagta gcctcgccga ccgaaagaac
     2521 gatggttttg gaggctagca tagacgtctt cctccttttg tgaggttgtc caccatcgtt
     2581 ctcgtagctg tgccgcctcc gcgacatggt ggcaagctta agttggatcc ccctggggag
     2641 agaggtcggt gattcggtca acgagggagc cgactgccga cgtgcgctcc ggaggcttgc
     2701 agaatgcgga acaccgcgcg ggcaggaaca gggcccacac taccgcccca caccccgcct
     2761 cccgcaccgc cccttcccgg ccgctgctct cggcgcgccc tgctgagcag ccgctattgg
     2821 ccacagccca tcgcggtcgg cgcgctgcca ttgctccctg gcgctgtccg tctgcgaggg
     2881 tactagtaag acgtgcggct tccgtttgtc acgtccggca cgccgcgaac cgcaaggaac
     2941 cttcccgact taggggcgga gcaggaagcg tcgccggggg gcccacaagg gtagcggcga
     3001 agatccgggt gacgctgcga acggacgtga agaatgtgcg agacccaggg tcggcgccgc
     3061 tgcgtttccc ggaaccacgc ccagagcagc cgcgtccctg cgcaaaccca gggctgcctt
     3121 ggaaaaggcg caacccgggc cccctcgagc cggcgccaaa gtggatctct gctgtccctg
     3181 taataaaccc gaaaattttg aatttttgta atttgttttt gtaattcttt agtttgtatg
     3241 tctgttgcta ttatgtctac tattctttcc cctgcactgt accccccaat cccccctttt
     3301 cttttaaaat tgtggatgaa tactgccatt tgtctcaaga tctagaattc aaaaaaagca
     3361 ccgactcggt gccacttttt caagttgata acggactagc cttatttaaa cttgctatgc
     3421 tgcgaatacg agcagaccct gatggtgtct gatacgtacc ctgatggtgt acgagaccac
     3481 ggagcccata tgctctcgtg gagaggagcg acgccatatc gtctgctccc tcgtattcgc
     3541 agcatagctc ttaaacagag acgcaaaaaa ccggtcgtct ccggtgtttc gtcctttcca
     3601 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     3661 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     3721 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac
     3781 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct ctctgggtcc
     3841 cctcggggtt gggaggtggg tctgaaacga taatggtgaa tatccctgcc taactctatt
     3901 cactatagaa agtacagcaa aaactattct taaacctacc aagcctccta ctatcattat
     3961 gaataatttt atataccaca gccaatttgt tatgttaaac caattccaca aacttgccca
     4021 tttatctaat tccaataatt cttgttcatt cttttcttgc tggttttgcg attcttcaat
     4081 taaggagtgt attaagcttg tgtaattgtt aatttctctg tcccactcca tccaggtcgt
     4141 gtgattccaa atctgttcca gagatttatt actccaacta gcattccaag gcacagcagt
     4201 ggtgcaaatg agttttccag agcaacccca aatccccagg agctgttgat cctttaggta
     4261 tctttccaca gccaggattc ttgcctggag ctgcttgatg ccccagactg tgagttgcaa
     4321 cagatgctgt tgcgcctcaa tagccctcag caaattgttc tgctgctgca ctataccaga
     4381 caataattgt ctggcctgta ccgtcagcgt cattgacgct gcgcccatag tgcttcctgc
     4441 tgctcccaag aacccaagga acaaagctcc tattcccact gctctttttt ctctctgcac
     4501 cactcttctc tttgccttgg tgggtgctac tcctaatggt tcaattttta ctactttata
     4561 tttatataat tcacttctcc aattgtccct catatctcct cctccaggtc tgaagatcag
     4621 cggccgcttg ctgtgcggtg gtcttacttt tgttttgctc ttcctctatc ttgtctaaag
     4681 cttccttggt gtcttttatc tctatccttt gatgcacaca atagagggtt gctactgtat
     4741 tatataatga tctaagttct tctgatcctg tctgaaggga tggttgtagc tgtcccagta
     4801 tttgtctaca gccttctgat gtttctaaca ggccaggatt aactgcgaat cgttctagct
     4861 ccctgcttgc ccatactata tgttttaatt tatatttttt ctttccccct ggccttaacc
     4921 gaattttttc ccatcgcgat ctaattctcc cccgcttaat actgacgctc tcgcacccat
     4981 ctctctcctt ctagcctccg ctagtcaaaa tttttggcgt actcaccagt cgccgcccct
     5041 cgcctcttgc cgtgcgcgct tcagcaagcc gagtcctgcg tcgagagagc tcctctggtt
     5101 tccctttcgc tttcaagtcc ctgttcgggc gccactgcta gagattttcc acactgacta
     5161 aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac acactacttg
     5221 aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt agccagagag
     5281 ctcccaggct cagatctggt ctaaccagag agacccgttt atgtatcgag ctaggcactt
     5341 aaatacaata tctctgcaat gcggcaattc agtggttcgt ccaatccatg tcagacccgt
     5401 ctgttgcctt cctaataagg cacgatcgta ccaccttact tccaccaatc ggcatgcacg
     5461 gtgctttttc tctccttgta aggcatgttg ctaactcatc gttaccatgt tgcaagacta
     5521 caagagtatt gcataagact acattaagct tgcagctcca gcttttgttc cctttagtga
     5581 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5641 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5701 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5761 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5821 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5881 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5941 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6001 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6061 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6121 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6181 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6241 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6301 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6361 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6421 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6481 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6541 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6601 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6661 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6721 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6781 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6841 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6901 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6961 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     7021 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7081 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7141 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7201 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7261 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7321 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7381 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7441 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7501 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7561 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7621 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7681 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7741 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     7801 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     7861 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     7921 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     7981 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     8041 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     8101 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     8161 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     8221 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     8281 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     8341 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     8401 agggcgaatt gggtacgtcc ctaggcctcc aaaaaagcct cctcactact tctggaatag
     8461 ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg
     8521 cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggat
     8581 agctagagcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     8641 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaa
//
