LOCUS       sequence-181743-        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"
     primer_bind     complement(6..23)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(177..765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(257..276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(936..1727,1728..1796))
                     /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     1559..1578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1797..1901)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1927..2382)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2064..2085)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2276..2295
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2509..2531
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2523..2540
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2524..2540
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2550..2569
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2550..2568
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2589..2724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2764..2885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2775..2794)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2829..2848
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2963..3196
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(3286..3882)
                     /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(3366..3386)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3863..3882
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(3910..3963)
                     /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             complement(3985..4134)
                     /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             complement(4159..4179)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4229..4253
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(4718..5228)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4781..4799)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    5277..5394
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(5668..5916)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5726..5745)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5896..5916)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(6094..6138)
                     /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    6323..6556
                     /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    7049..7174
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             7221..7401
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7402..7628)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(7656..7676)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7656..7674)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7695..7711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7695..7711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7708..7730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7719..7735
                     /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)."
     promoter        complement(join(7743..7749,7750..7767,7768..7773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7788..7809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
       61 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      121 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      181 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      241 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      301 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      361 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      421 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      481 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      541 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      601 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      661 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      721 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      781 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      841 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      901 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      961 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1021 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1081 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1141 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1201 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1261 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1321 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1381 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1441 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1501 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1561 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1621 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1681 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1741 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1801 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1861 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1921 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1981 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2041 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2101 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2161 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2221 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2281 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2341 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     2401 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     2461 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     2521 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
     2581 cgtccctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc
     2641 ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga
     2701 actgggcgga gttaggggcg ggatgggcgg agttaggggc gggatagcta gagccagaca
     2761 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     2821 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2881 aagttcctct cactctctga tattcatttc tttgcaagtt ataaatactg aataataaga
     2941 tgacatgaac tactatacgt actgctagag attttccaca ctgactaaaa gggtctgagg
     3001 gatctctagt taccagagtc acacaacaga cgggcacaca ctacttgaag cactcaaggc
     3061 aagctttatt gaggcttaag cagtgggttc cctagttagc cagagagctc ccaggctcag
     3121 atctggtcta accagagaga cccagtacaa gcaaaaagca gatcttgtct tcgttgggag
     3181 tgaattagcc cttccagtcc ccccttttct tttaaaaagt ggctaagatc tacagctgcc
     3241 ttgtaagtca ttggtcttaa agtcgactta acgcgttcag ctagcggcac cgggcttgcg
     3301 ggtcatgcac caggtgcgcg gtccttcggg cacctcgacg tcggcggtga cggtgaagcc
     3361 gagccgctcg tagaagggga ggttgcgggg cgcggaggtc tccaggaagg cgggcacccc
     3421 ggcgcgctcg gccgcctcca ctccggggag cacgacggcg ctgcccagac ccttgccctg
     3481 gtggtcgggc gaaacgccga cggtggccag gaaccacgcg ggctccttgg gccggtgcgg
     3541 cgccaggagg ccttccatct gttgctgcgc ggccagccgg gaaccgctca actcggccat
     3601 gcgcgggccg atctcggcga acaccgcccc cgcttcgacg ctctccggcg tggtccagac
     3661 cgccacggcg gcgccgtcgt ccgcgaccca caccttgccg atgtcgagcc cgacgcgcgt
     3721 gaggaagagt tcttgcagct cggtgacccg ctcgatgtgg cggtccggat cgacggtgtg
     3781 gcgcgtggcg gggtagtcgg cgaacgcggc ggcgagggtg cgtacggccc tggggacgtc
     3841 gtcgcgggtg gcgaggcgca ccgtgggctt gtactcggtc atggtaagct ccggtggatc
     3901 tgtggtacct gggccaggat tctcctcgac gtcaccgcat gttagcagac ttcctctgcc
     3961 ctctccactg ccgatatcgt taatcagcat gtccaggtcg aaatcatcaa gggcgtcact
     4021 gccgagcatg tcgaggtcaa agtcatcaag ggcatccgaa ccaagcatgt caaggtcaaa
     4081 atcatcgagg gcgtcacttc ccagcatatc cagatcaaaa tcgtccaatg cgtcagcccg
     4141 tccggtacca gcggccgcca ccttcctctt tttcttaggt ccgctacctc ctcctccgct
     4201 tcctcctcct ccgcttccac ctcctccgcc cactttgtac aagaaagttg aagaacggcc
     4261 cagcggcaca aggttgacga caagatcttc gacctgcgag gtcgcgacga gggacttggt
     4321 caaatcgtac aacgatttgc gcgaggcctc ggtgctattc gcaacgattg tcacgtcgtg
     4381 cgaccatacc tgagtgtagc gcactttcgg aagtccggaa tcaacgacgt ccgcctgatc
     4441 cagttttagg ttgactcgat acgcggtctt ggctccgttt tgacggagcg aagccgtgag
     4501 gcgcagccga cccaccagag gcccgacctt ctcttcgaag atctgacggt ctgcggtgga
     4561 ctggatctca gtcagagtgc gagtagcctc gccgaccgaa agaacgatgg ttttggaggc
     4621 tagcatagac gtcttcctcc ttttgtgagg ttgtccacca tcgttctcgt agctgtgccg
     4681 cctccgcgac atggtggcaa gcttaagttg gatccccctg gggagagagg tcggtgattc
     4741 ggtcaacgag ggagccgact gccgacgtgc gctccggagg cttgcagaat gcggaacacc
     4801 gcgcgggcag gaacagggcc cacactaccg ccccacaccc cgcctcccgc accgcccctt
     4861 cccggccgct gctctcggcg cgccctgctg agcagccgct attggccaca gcccatcgcg
     4921 gtcggcgcgc tgccattgct ccctggcgct gtccgtctgc gagggtacta gtaagacgtg
     4981 cggcttccgt ttgtcacgtc cggcacgccg cgaaccgcaa ggaaccttcc cgacttaggg
     5041 gcggagcagg aagcgtcgcc ggggggccca caagggtagc ggcgaagatc cgggtgacgc
     5101 tgcgaacgga cgtgaagaat gtgcgagacc cagggtcggc gccgctgcgt ttcccggaac
     5161 cacgcccaga gcagccgcgt ccctgcgcaa acccagggct gccttggaaa aggcgcaacc
     5221 cgggccccct cgagccggcg ccaaagtgga tctctgctgt ccctgtaata aacccgaaaa
     5281 ttttgaattt ttgtaatttg tttttgtaat tctttagttt gtatgtctgt tgctattatg
     5341 tctactattc tttcccctgc actgtacccc ccaatccccc cttttctttt aaaattgtgg
     5401 atgaatactg ccatttgtct caagatctag aattcaaaaa aagcaccgac tcggtgccac
     5461 tttttcaagt tgataacgga ctagccttat ttaaacttgc tatgctgcga atacgagcag
     5521 accctgatgg tgtctgatac gtaccctgat ggtgtacgag accacggagc ccatatgctc
     5581 tcgtggagag gagcgacgcc atatcgtctg ctccctcgta ttcgcagcat agctcttaaa
     5641 cagagacgca aaaaaccggt cgtctccggt gtttcgtcct ttccacaaga tatataaagc
     5701 caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa
     5761 ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa
     5821 tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat
     5881 atgcaaatat gaaggaatca tgggaaatag gccctctctg ggtcccctcg gggttgggag
     5941 gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta tagaaagtac
     6001 agcaaaaact attcttaaac ctaccaagcc tcctactatc attatgaata attttatata
     6061 ccacagccaa tttgttatgt taaaccaatt ccacaaactt gcccatttat ctaattccaa
     6121 taattcttgt tcattctttt cttgctggtt ttgcgattct tcaattaagg agtgtattaa
     6181 gcttgtgtaa ttgttaattt ctctgtccca ctccatccag gtcgtgtgat tccaaatctg
     6241 ttccagagat ttattactcc aactagcatt ccaaggcaca gcagtggtgc aaatgagttt
     6301 tccagagcaa ccccaaatcc ccaggagctg ttgatccttt aggtatcttt ccacagccag
     6361 gattcttgcc tggagctgct tgatgcccca gactgtgagt tgcaacagat gctgttgcgc
     6421 ctcaatagcc ctcagcaaat tgttctgctg ctgcactata ccagacaata attgtctggc
     6481 ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt cctgctgctc ccaagaaccc
     6541 aaggaacaaa gctcctattc ccactgctct tttttctctc tgcaccactc ttctctttgc
     6601 cttggtgggt gctactccta atggttcaat ttttactact ttatatttat ataattcact
     6661 tctccaattg tccctcatat ctcctcctcc aggtctgaag atcagcggcc gcttgctgtg
     6721 cggtggtctt acttttgttt tgctcttcct ctatcttgtc taaagcttcc ttggtgtctt
     6781 ttatctctat cctttgatgc acacaataga gggttgctac tgtattatat aatgatctaa
     6841 gttcttctga tcctgtctga agggatggtt gtagctgtcc cagtatttgt ctacagcctt
     6901 ctgatgtttc taacaggcca ggattaactg cgaatcgttc tagctccctg cttgcccata
     6961 ctatatgttt taatttatat tttttctttc cccctggcct taaccgaatt ttttcccatc
     7021 gcgatctaat tctcccccgc ttaatactga cgctctcgca cccatctctc tccttctagc
     7081 ctccgctagt caaaattttt ggcgtactca ccagtcgccg cccctcgcct cttgccgtgc
     7141 gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc tggtttccct ttcgctttca
     7201 agtccctgtt cgggcgccac tgctagagat tttccacact gactaaaagg gtctgaggga
     7261 tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa
     7321 gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat
     7381 ctggtctaac cagagagacc cgtttatgta tcgagctagg cacttaaata caatatctct
     7441 gcaatgcggc aattcagtgg ttcgtccaat ccatgtcaga cccgtctgtt gccttcctaa
     7501 taaggcacga tcgtaccacc ttacttccac caatcggcat gcacggtgct ttttctctcc
     7561 ttgtaaggca tgttgctaac tcatcgttac catgttgcaa gactacaaga gtattgcata
     7621 agactacatt aagcttgcag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     7681 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     7741 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     7801 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     7861 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
//
