LOCUS       sequence-389696-        8283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning pairs of AsCas12a guides with two DRs that are as 
            different as possible. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8283)
  TITLE     Schmierer unpublished plasmids
REFERENCE   2  (bases 1 to 8283)
  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"
COMMENT     Sequence Label: pRDA52_Lenti.puro.U6_modified DR
FEATURES             Location/Qualifiers
     source          1..8283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1719..1967
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1719..1739
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1890..1909
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2074..2191
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2330..3508
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2561..3499
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3456..3476
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3521..4120
                     /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(3521..3540)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4017..4037
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4136..4724
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4189..4209)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4607..4618)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4796..5029
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5107..5228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5144..5163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5198..5217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5268..5403
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5423..5442)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5424..5442)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5452..5469)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5452..5468)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5461..5483)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5610..6065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5697..5716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5907..5928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6091..6195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6196..6264,6265..7056)
                     /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(6414..6433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7227..7815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7716..7735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7969..7986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8103..8124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8139..8144,8145..8162,8163..8169)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8177..8193
                     /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     8182..8204
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8201..8217
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8201..8217
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8236..8256
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8238..8256
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gaggtaccga gggcctattt cccatgattc
     1741 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     1801 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1861 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1921 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgaa atttctcctc
     1981 ttggagatgg agacgcgttt tttctcgtct ctgaattcgc tagcaagctt ggcgtaacta
     2041 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2101 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2161 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2221 actttggcgc cggctcgagg gggcccgggt gcaaagatgg ataaagtttt aaacagagag
     2281 gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc
     2341 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2401 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2461 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2521 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     2581 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg
     2641 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     2701 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     2761 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     2821 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     2881 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     2941 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     3001 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     3061 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     3121 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     3181 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     3241 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     3301 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     3361 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     3421 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     3481 tttttttctt ccatttcagg tgtcgtgacg tacggccacc atgaccgagt acaagcccac
     3541 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3601 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3661 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3721 ggacgacggc gccgccgtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3781 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3841 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3901 cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3961 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     4021 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     4081 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca
     4141 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     4201 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     4261 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     4321 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     4381 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     4441 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     4501 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     4561 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     4621 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     4681 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca
     4741 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     4801 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     4861 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4921 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4981 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt
     5041 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     5101 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5161 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5221 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
     5281 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     5341 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     5401 gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     5461 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5521 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5581 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     5641 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5701 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5761 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5821 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5881 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5941 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     6001 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     6061 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     6121 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     6181 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     6241 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     6301 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     6361 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     6421 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6481 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6541 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6601 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6661 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6721 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6781 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6841 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6901 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6961 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     7021 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     7081 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     7141 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7201 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     7261 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7321 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     7381 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7441 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7501 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7561 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7621 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7681 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7741 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7801 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7861 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7921 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7981 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     8041 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     8101 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     8161 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     8221 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
     8281 taa
//
