LOCUS       sequence-326103-        8322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiGuide-Puro backbone with cloned target sequence for human 
            PRMT5. The insert is: 5´- CACCGAGCCGCGTGTCCAGCGGGA-3. sgRNA achieves
            a downregulation of PRMT5 in combination with dCAS9-KRAB-MCP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8322)
  TITLE     sgRNA MS2 (zeo_backbone) target c-MYC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8322)
  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: 
            "Unpublished"
COMMENT     Sequence Label: lentiGuide-Puro PRMT5-CRISPRi
FEATURES             Location/Qualifiers
     source          1..8322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    592..717
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             764..944
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(945..1171)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(1199..1219)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1199..1217)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1238..1254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1238..1254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1251..1273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1262..1278
                     /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(1286..1292,1293..1310,1311..1316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1331..1352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1469..1486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1640..2228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1720..1739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2399..3190,3191..3259))
                     /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     3022..3041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3260..3364)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3390..3845)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3527..3548)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3739..3758
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3972..3994
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3986..4003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3987..4003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4013..4032
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4013..4031
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      4052..4187
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4227..4348
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4238..4257)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4292..4311
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             4426..4659
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    4731..5319
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4837..4848
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     5246..5266
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5335..5934)
                     /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(5418..5438)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5915..5934
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5947..7125)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5956..6894
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(5979..5999)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    7264..7381
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(7437..7512)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7533..7781)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7591..7610)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7761..7781)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(7959..8003)
                     /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    join(8188..8322,1..99)
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 ataccagaca ataattgtct ggcctgtacc gtcagcgtca ttgacgctgc gcccatagtg
       61 cttcctgctg ctcccaagaa cccaaggaac aaagctccta ttcccactgc tcttttttct
      121 ctctgcacca ctcttctctt tgccttggtg ggtgctactc ctaatggttc aatttttact
      181 actttatatt tatataattc acttctccaa ttgtccctca tatctcctcc tccaggtctg
      241 aagatcagcg gccgcttgct gtgcggtggt cttacttttg ttttgctctt cctctatctt
      301 gtctaaagct tccttggtgt cttttatctc tatcctttga tgcacacaat agagggttgc
      361 tactgtatta tataatgatc taagttcttc tgatcctgtc tgaagggatg gttgtagctg
      421 tcccagtatt tgtctacagc cttctgatgt ttctaacagg ccaggattaa ctgcgaatcg
      481 ttctagctcc ctgcttgccc atactatatg ttttaattta tattttttct ttccccctgg
      541 ccttaaccga attttttccc atcgcgatct aattctcccc cgcttaatac tgacgctctc
      601 gcacccatct ctctccttct agcctccgct agtcaaaatt tttggcgtac tcaccagtcg
      661 ccgcccctcg cctcttgccg tgcgcgcttc agcaagccga gtcctgcgtc gagagagctc
      721 ctctggtttc cctttcgctt tcaagtccct gttcgggcgc cactgctaga gattttccac
      781 actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac
      841 actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag
      901 ccagagagct cccaggctca gatctggtct aaccagagag acccgtttat gtatcgagct
      961 aggcacttaa atacaatatc tctgcaatgc ggcaattcag tggttcgtcc aatccatgtc
     1021 agacccgtct gttgccttcc taataaggca cgatcgtacc accttacttc caccaatcgg
     1081 catgcacggt gctttttctc tccttgtaag gcatgttgct aactcatcgt taccatgttg
     1141 caagactaca agagtattgc ataagactac attaagcttg cagctccagc ttttgttccc
     1201 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1261 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1321 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1381 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1441 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1501 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1561 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1801 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2041 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2281 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2341 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2401 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2461 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2521 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2581 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2641 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2701 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2761 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2821 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2881 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2941 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3001 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3061 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3361 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     3421 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3481 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3541 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3601 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3661 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3721 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3781 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3841 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     3901 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     3961 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga
     4021 ctcactatag ggcgaattgg gtacgtccct aggcctccaa aaaagcctcc tcactacttc
     4081 tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     4141 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     4201 ggcgggatag ctagagccag acatgataag atacattgat gagtttggac aaaccacaac
     4261 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4321 aaccattata agctgcaata aacaagttcc tctcactctc tgatattcat ttctttgcaa
     4381 gttataaata ctgaataata agatgacatg aactactata cgtactgcta gagattttcc
     4441 acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac
     4501 acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt
     4561 agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta caagcaaaaa
     4621 gcagatcttg tcttcgttgg gagtgaatta gcccttccag tccccccttt tcttttaaaa
     4681 agtggctaag atctacagct gccttgtaag tcattggtct taaagtcgac gcggggaggc
     4741 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     4801 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     4861 gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag gaaaggacga
     4921 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     4981 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     5041 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     5101 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     5161 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     5221 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     5281 agtcaatctt tcacaaattt tgtaatccag aggttgattg tcgacttaac gcgttcaggc
     5341 accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt
     5401 gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa
     5461 ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag
     5521 acccttgccc tggtggtcgg gcgagactcc gacggtggcc aggaaccacg cgggctcctt
     5581 gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct
     5641 caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg
     5701 cgtggtccag accgccacgg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag
     5761 cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg
     5821 atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc
     5881 cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatggtggc
     5941 cgtacgtcac gacacctgaa atggaagaaa aaaactttga accactgtct gaggcttgag
     6001 aatgaaccaa gatccaaact caaaaagggc aaattccaag gagaattaca tcaagtgcca
     6061 agctggccta acttcagtct ccacccactc agtgtgggga aactccatcg cataaaaccc
     6121 ctccccccaa cctaaagacg acgtactcca aaagctcgag aactaatcga ggtgcctgga
     6181 cggcgcccgg tactccgtgg agtcacatga agcgacggct gaggacggaa aggccctttt
     6241 cctttgtgtg ggtgactcac ccgcccgctc tcccgagcgc cgcgtcctcc attttgagct
     6301 ccctgcagca gggccgggaa gcggccatct ttccgctcac gcaactggtg ccgaccgggc
     6361 cagccttgcc gcccagggcg gggcgataca cggcggcgcg aggccaggca ccagagcagg
     6421 ccggccagct tgagactacc cccgtccgat tctcggtggc cgcgctcgca ggccccgcct
     6481 cgccgaacat gtgcgctggg acgcacgggc cccgtcgccg cccgcggccc caaaaaccga
     6541 aataccagtg tgcagatctt ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt
     6601 cgcagcaggt catcaaaaat tttaaatggc tagagactta tcgaaagcag cgagacaggc
     6661 gcgaaggtgc caccagattc gcacgcggcg gccccagcgc ccaggccagg cctcaactca
     6721 agcacgaggc gaaggggctc cttaagcgca aggcctcgaa ctctcccacc cacttccaac
     6781 ccgaagctcg ggatcaagaa tcacgtactg cagccaggtg gaagtaattc aaggcacgca
     6841 agggccataa cccgtaaaga ggccaggccc gcgggaacca cacacggcac ttacctgtgt
     6901 tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg
     6961 ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac
     7021 cccgcgccac cttctctagg caccggatca attgccgacc cctcccccca acttctcggg
     7081 gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagccaattc ccactccttt
     7141 caagacctag aaggtccatt agctgcaaag attcctctct gtttaaaact ttatccatct
     7201 ttgcacccgg gccccctcga gccggcgcca aagtggatct ctgctgtccc tgtaataaac
     7261 ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta tgtctgttgc
     7321 tattatgtct actattcttt cccctgcact gtacccccca atcccccctt ttcttttaaa
     7381 attgtggatg aatactgcca tttgtctcaa gatctagtta cgccaagctt aaaaaagcac
     7441 cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc
     7501 tagctctaaa actcccgctg gacacgcggc tcggtgtttc gtcctttcca caagatatat
     7561 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     7621 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     7681 actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac agccttgtat
     7741 cgtatatgca aatatgaagg aatcatggga aataggccct ctctgggtcc cctcggggtt
     7801 gggaggtggg tctgaaacga taatggtgaa tatccctgcc taactctatt cactatagaa
     7861 agtacagcaa aaactattct taaacctacc aagcctccta ctatcattat gaataatttt
     7921 atataccaca gccaatttgt tatgttaaac caattccaca aacttgccca tttatctaat
     7981 tccaataatt cttgttcatt cttttcttgc tggttttgcg attcttcaat taaggagtgt
     8041 attaagcttg tgtaattgtt aatttctctg tcccactcca tccaggtcgt gtgattccaa
     8101 atctgttcca gagatttatt actccaacta gcattccaag gcacagcagt ggtgcaaatg
     8161 agttttccag agcaacccca aatccccagg agctgttgat cctttaggta tctttccaca
     8221 gccaggattc ttgcctggag ctgcttgatg ccccagactg tgagttgcaa cagatgctgt
     8281 tgcgcctcaa tagccctcag caaattgttc tgctgctgca ct
//
