LOCUS       sequence-298306-        7646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression plasmid for two sgRNAs from a hU6 and a mU6 
            promoter. The cloning site design allows a one-step cloning strategy
            of both sgRNAs. Expresses a Thy1.1 marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7646)
  AUTHORS   Umkehrer C, Holstein F, Formenti L, Jude J, Froussios K, Neumann T, 
            Cronin SM, Haas L, Lipp JJ, Burkard TR, Fellner M, Wiesner T, Zuber 
            J, Obenauf AC
  TITLE     Isolating live cell clones from barcoded populations using 
            CRISPRa-inducible reporters.
  JOURNAL   Nat Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0.
  PUBMED    32719478
REFERENCE   2  (bases 1 to 7646)
  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 
            Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0."
COMMENT     Sequence Label: Dual-sgRNA_hU6-mU6
FEATURES             Location/Qualifiers
     source          1..7646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             105..285
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    332..457
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    950..1183
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1368..1412
                     /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        1590..1838
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1590..1610
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1761..1780
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2024..2141
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2185..2498
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2417..2437
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2646..2857
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2877..3365
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     misc_feature    3376..3964
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3429..3449)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3847..3858)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4036..4269
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4347..4468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4384..4403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4438..4457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4508..4643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4663..4682)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4664..4682)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4692..4709)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4692..4708)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4701..4723)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4850..5305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4937..4956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5147..5168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5331..5435
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5436..5504,5505..6296)
                     /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(5654..5673)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6467..7055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6956..6975
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7209..7226
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7343..7364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7379..7384,7385..7402,7403..7409)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7417..7433
                     /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     7422..7444
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7441..7457
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7441..7457
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7476..7496
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7478..7496
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        join(7524..7646,1..104)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
       61 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
      121 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
      181 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
      241 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
      301 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
      361 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
      421 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
      481 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
      541 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
      601 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
      661 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
      721 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
      781 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
      841 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
      901 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
      961 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     1021 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     1081 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     1141 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     1201 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     1261 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     1321 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     1381 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     1441 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     1501 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     1561 cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt ccttcatatt
     1621 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     1681 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     1741 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     1801 tcttggcttt atatatcttg tggaaaggac gaaacaccgg agacgtttca tgtcttctaa
     1861 cttcgaaatc gatgtagtct tccgtctctg tttaagagct atgctggaaa cagcatagca
     1921 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1981 ttgaattcta gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2041 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2101 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2161 gcagagatcc actttggcgc cggcgatccg acgccgccat ctctaggccc gcgccggccc
     2221 cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa tttgcatata
     2281 atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat ctgttctttt
     2341 taatactagc tacattttac atgataggct tggatttcta taagagatac aaatactaaa
     2401 ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt aattgtgtgt
     2461 tttgagacta taaatatccc ttggagaaaa gccttgttgt cttccaccgg ggacgagtgt
     2521 ctaagaagac taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     2581 tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tcgagtggct ccggtgcccg
     2641 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2701 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2761 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2821 cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg gccaccatga
     2881 acccagccat cagcgtcgct ctcctgctct cagtcttgca ggtgtcccga gggcagaagg
     2941 tgaccagcct gacagcctgc ctggtgaacc aaaaccttcg cctggactgc cgccatgaga
     3001 ataacaccaa ggataactcc atccagcatg agttcagcct gacccgagag aagaggaagc
     3061 acgtgctctc aggcaccctc gggatacccg agcacacgta ccgctcccgc gtcaccctct
     3121 ccaaccagcc ctatatcaag gtccttaccc tagccaactt caccaccaag gatgagggcg
     3181 actacttttg tgagcttcga gtctcgggcg cgaatcccat gagctccaac aaaagtatca
     3241 gtgtgtatag agacaaactg gtcaagtgtg gcggcataag cctgctggtt cagaacacat
     3301 cctggatgct gctgctgctg ctttccctct ccctcctcca agccctggac ttcatttctc
     3361 tgtgattaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3421 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3481 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3541 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3601 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3661 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3721 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3781 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3841 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3901 cctcttccgc gtctacgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3961 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     4021 aagaaaaggg gggactggaa gggctaattc actcccaacg aagtcaagat ctgctttttg
     4081 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4141 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4201 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4261 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     4321 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     4381 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4441 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     4501 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4561 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4621 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     4681 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4741 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4801 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4861 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4921 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4981 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5041 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5101 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5161 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5221 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5281 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5341 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5401 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5461 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5521 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5581 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5641 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5701 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5761 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5821 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5881 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5941 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6001 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6061 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6121 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6181 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6241 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6301 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6361 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6421 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6481 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6541 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6601 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6661 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6721 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6781 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6841 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6901 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6961 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7021 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7081 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7141 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7201 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7261 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7321 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7381 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7441 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     7501 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
     7561 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
     7621 ggtggaagta aggtggtacg atcgtg
//
