LOCUS       sequence-362663-        7463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector that enables constitutive sgRNA expression. Also 
            encodes Thy1.1 as a marker of transduction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  AUTHORS   Milling LE, Markson SC, Tjokrosurjo Q, Derosia NM, Streeter ISL, 
            Hickok GH, Lemmen AM, Nguyen TH, Prathima P, Fithian W, Schwartz MA,
            Hacohen N, Doench JG, LaFleur MW, Sharpe AH
  TITLE     Framework for in vivo T cell screens.
  JOURNAL   J Exp Med. 2024 Apr 1;221(4):e20230699. doi: 10.1084/jem.20230699. 
            Epub 2024 Feb 27.
  PUBMED    38411617
REFERENCE   2  (bases 1 to 7463)
  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: "J Exp 
            Med."; date: "2024-04-1"; volume: "221(4):e20230699. doi"; pages: " 
            10.1084/jem.20230699. Epub 2024 Feb 27"
COMMENT     Sequence Label: pRDA_526
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     214..232
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    804..1392
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(857..877)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1275..1286)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1464..1697
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1775..1896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1812..1831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1866..1885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1936..2071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2091..2110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2092..2110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2120..2137)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2120..2136)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2129..2151)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2278..2733
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2365..2384)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2575..2596
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2759..2863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2864..2932,2933..3724)
                     /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(3082..3101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3895..4483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4384..4403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4637..4654
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4771..4792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4807..4812,4813..4830,4831..4837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4845..4861
                     /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     4850..4872
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4869..4885
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4869..4885
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4904..4924
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4906..4924
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4952..5178
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5179..5359
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5406..5531
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6024..6257
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6442..6486
                     /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        6667..6915
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6667..6687
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6838..6857
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7082..7199
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7248..7463,1..295)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 cgcggcgtgc cggacgtgac aaacggaagc cgcacatctc actagtaccc tcgcagacgg
       61 acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg
      121 ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg
      181 ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga
      241 gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggat
      301 ccgccaccat gaaccctgct attagtgtag cacttttact ttctgtgctg caggtgagca
      361 ggggccagaa ggtgaccagc ttgacagcct gcctggtcaa ccagaatctc cgccttgact
      421 gccggcatga aaacaatacc aaggacaaca gcatccagca cgagttttca ctcactcggg
      481 agaagaggaa acatgtgttg tccggtacgc tgggcattcc agagcacacc tatcgaagtc
      541 gtgtcaccct cagcaatcaa ccgtacatca aagttctgac tctggccaac tttactacaa
      601 aagatgaagg ggattacttc tgtgaactga gagtgtcagg agctaatccc atgtcctcaa
      661 ataaatccat aagtgtctat agagataagc tggtgaagtg tgggggaatt tccttgctag
      721 ttcagaacac atcgtggatg ctgctgttac tcctctcttt gtctctgctc caagcactgg
      781 acttcatctc tctatgacgt acgaatcaac ctctggatta caaaatttgt gaaagattga
      841 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
      901 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
      961 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     1021 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     1081 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     1141 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     1201 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     1261 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     1321 ctctgcggcc tcttccgcgt ctttgccttc gccctcagac gagtcggatc tccctttggg
     1381 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     1441 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     1501 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     1561 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     1621 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     1681 gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata
     1741 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     1801 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1861 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     1921 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     1981 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2041 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     2101 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2161 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2221 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     2281 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2341 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2401 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2461 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2521 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2581 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2641 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2701 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2761 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2821 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2881 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2941 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3001 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3061 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3121 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3181 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3241 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3301 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3361 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3421 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3481 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3541 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3601 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3661 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3721 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3781 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3841 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3901 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3961 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4021 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4081 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4141 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4201 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4261 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4321 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4441 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4501 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4561 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4621 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4681 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4741 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4801 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4861 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     4921 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
     4981 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
     5041 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
     5101 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
     5161 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5221 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5281 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5341 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     5401 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     5461 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     5521 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     5581 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     5641 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     5701 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     5761 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     5821 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     5881 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     5941 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     6001 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     6061 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     6121 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     6181 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     6241 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     6301 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     6361 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     6421 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     6481 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     6541 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     6601 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     6661 ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     6721 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     6781 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     6841 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     6901 aaaggacgaa acaccggaga cgcttttttg tttgagagct agaaacgtct catagcaagt
     6961 tcaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcgctcacct
     7021 attagcggct aaggtttttt gaattctcga cctcgagaca aatggcagta ttcatccaca
     7081 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     7141 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     7201 gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg gcccgggttg
     7261 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     7321 gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc
     7381 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     7441 agtcgggaag gttccttgcg gtt
//
