LOCUS       sequence-178377-        7920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for CRISPRa, lentiviral expression of gRNA scaffold using activator 
            VP64.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7920)
  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 7920)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-12-21"; volume: "9(1):5416. doi"; pages: " 
            10.1038/s41467-018-07901-8"
COMMENT     Sequence Label: pXPR_501
FEATURES             Location/Qualifiers
     source          1..7920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2239..2356
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2405..2915
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2834..2852
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    complement(3380..3404)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3454..3474
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3499..3648
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             3670..3723
                     /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             3751..4347
                     /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(3751..3770)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4247..4267
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4437..4670
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4748..4869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4785..4804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4839..4858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4909..5044
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5064..5083)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5065..5083)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5093..5110)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5093..5109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5102..5124)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5251..5706
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5338..5357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5548..5569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5732..5836
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5837..5905,5906..6697)
                     /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     complement(6055..6074)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6868..7456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7357..7376
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7610..7627
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7744..7765
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7780..7785,7786..7803,7804..7810)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7818..7834
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     7823..7845
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7842..7858
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7842..7858
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7877..7897
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7879..7897
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgaccggttt
     1981 tttgcgtctc tgtttaagag ctatgctgcg aatacgaggg agcagacgat atggcgtcgc
     2041 tcctctccac gagagcatat gggctccgtg gtctcgtaca ccatcagggt acgtatcaga
     2101 caccatcagg gtctgctcgt attcgcagca tagcaagttt aaataaggct agtccgttat
     2161 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct tgagacaaat
     2221 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2281 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2341 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct
     2401 cgagggggcc cgggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct
     2461 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca
     2521 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc
     2581 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac
     2641 aaacggaagc cgcacgtctt actagtaccc tcgcagacgg acagcgccag ggagcaatgg
     2701 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga
     2761 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt
     2821 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct
     2881 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccaacttaa gcttgccacc
     2941 atgtcgcgga ggcggcacag ctacgagaac gatggtggac aacctcacaa aaggaggaag
     3001 acgtctatgc tagcctccaa aaccatcgtt ctttcggtcg gcgaggctac tcgcactctg
     3061 actgagatcc agtccaccgc agaccgtcag atcttcgaag agaaggtcgg gcctctggtg
     3121 ggtcggctgc gcctcacggc ttcgctccgt caaaacggag ccaagaccgc gtatcgagtc
     3181 aacctaaaac tggatcaggc ggacgtcgtt gattccggac ttccgaaagt gcgctacact
     3241 caggtatggt cgcacgacgt gacaatcgtt gcgaatagca ccgaggcctc gcgcaaatcg
     3301 ttgtacgatt tgaccaagtc cctcgtcgcg acctcgcagg tcgaagatct tgtcgtcaac
     3361 cttgtgccgc tgggccgttc ttcaactttc ttgtacaaag tgggcggagg aggtggaagc
     3421 ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa ggtggcggcc
     3481 gctggtaccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac
     3541 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc
     3601 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taacgatatc
     3661 ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
     3721 ccaggtacca cagatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc
     3781 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3841 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3901 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3961 gccgccgtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4021 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4081 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtt
     4141 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4201 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4261 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     4321 tggtgcatga cccgcaagcc cggtgccgct agctgaacgc gttaagtcga ctttaagacc
     4381 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4441 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4501 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4561 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4621 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag
     4681 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     4741 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4801 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4861 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
     4921 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4981 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5041 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     5101 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5161 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5221 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5281 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5341 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5401 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5461 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5521 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5581 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5641 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5701 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5761 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5821 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5881 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5941 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6001 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6061 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6121 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6181 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6241 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6301 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6361 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6421 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6481 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6541 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6601 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6661 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6721 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6781 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6841 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6901 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6961 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7021 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7081 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7141 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7201 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7261 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7321 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7381 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7441 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7501 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7561 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     7621 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     7681 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     7741 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     7801 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     7861 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
//
