LOCUS       sequence-330194-        7679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Higher titer version of pXPR_053. Lentiviral vector that enables 
            constitutive sgRNA expression. Also encodes the fluorophore 
            violet-excited GFP (Vex) as a marker of transduction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7679)
  AUTHORS   LaFleur MW, Lemmen AM, Streeter ISL, Nguyen TH, Milling LE, Derosia 
            NM, Hoffman ZM, Gillis JE, Tjokrosurjo Q, Markson SC, Huang AY, 
            Anekal PV, Montero Llopis P, Haining WN, Doench JG, Sharpe AH
  TITLE     X-CHIME enables combinatorial, inducible, lineage-specific and 
            sequential knockout of genes in the immune system.
  JOURNAL   Nat Immunol. 2023 Nov 27. doi: 10.1038/s41590-023-01689-6.
  PUBMED    38012416
REFERENCE   2  (bases 1 to 7679)
  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 
            Immunol. 2023 Nov 27. doi: 10.1038/s41590-023-01689-6."
COMMENT     Sequence Label: pXPR_071
FEATURES             Location/Qualifiers
     source          1..7679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             66..299
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    377..498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(414..433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     468..487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      538..673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(693..712)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(694..712)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(722..739)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(722..738)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(731..753)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      880..1335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(967..986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1177..1198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1361..1465
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1466..1534,1535..2326)
                     /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(1684..1703)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2497..3085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2986..3005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3239..3256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3373..3394
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3409..3414,3415..3432,3433..3439)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3447..3463
                     /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     3452..3474
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3471..3487
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3471..3487
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3506..3526
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3508..3526
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3554..3780
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3781..3961
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4008..4133
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4626..4859
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5044..5088
                     /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        5266..5514
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5266..5286
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5437..5456
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    5669..5786
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5835..6345
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6264..6282
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      6353..6362
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    7085..7673
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7138..7158)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7556..7567)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
       61 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
      121 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      181 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      241 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      301 tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
      361 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
      421 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
      481 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
      541 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
      601 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
      661 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
      721 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
      781 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
      841 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
      901 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      961 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1021 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1081 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1141 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1201 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1261 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1321 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1381 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1441 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1501 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1561 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1621 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1681 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1741 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1801 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1861 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1921 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1981 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     2041 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2101 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2161 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2221 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2281 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2341 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2401 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2461 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2521 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2581 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2641 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2701 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2761 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2821 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2881 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2941 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3001 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3061 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3121 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3181 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3241 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     3301 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     3361 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     3421 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     3481 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
     3541 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
     3601 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
     3661 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
     3721 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
     3781 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     3841 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     3901 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     3961 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
     4021 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
     4081 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
     4141 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
     4201 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     4261 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     4321 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     4381 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     4441 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
     4501 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     4561 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     4621 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     4681 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     4741 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     4801 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     4861 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     4921 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     4981 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     5041 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     5101 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     5161 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     5221 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc
     5281 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     5341 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     5401 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     5461 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac
     5521 ggtcgtctct gtttcagagc tatgctggaa actgcatagc aagttgaaat aaggctagtc
     5581 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttatgcggaa ttctcgacct
     5641 cgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     5701 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     5761 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt
     5821 ggccgcggct cgagggggcc cgggttgcgc cttttccaag gcagccctgg gtttgcgcag
     5881 ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc
     5941 acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc
     6001 ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc
     6061 cggacgtgac aaacggaagc cgcacatctc actagtaccc tcgcagacgg acagcgccag
     6121 ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg
     6181 cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg
     6241 tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg
     6301 gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggat ccgccaccat
     6361 ggtctccaaa ggcgaggagc tgtttacagg cgttgttcct attttggtgg agttggatgg
     6421 agatgtcaac gggcataaat tcagcgttag tggggagggt gagggagatg ctacttatgg
     6481 gaaactcacc ctcaaattta tctgcactac tggcaagctt cccgtccctt ggcctaccct
     6541 ggtaactaca ttttcctatg gtgtacagtg cttctccaga tacccagatc acatgaagca
     6601 acatgatttc tttaagtctg ccatgccaga gggatacgtt caggaacgca ccatttcatt
     6661 caaggacgac ggaaactata agactcgcgc agaggtaaag ttcgagggag atacactcgt
     6721 aaacaggatc gaactgaagg ggatagattt taaggaggac ggtaatatac tcgggcataa
     6781 actcgagtat aactacaact cccataacgt ctatattacc gccgataaac agaagaacgg
     6841 cattaaggcc aatttcaaaa taagacacaa tatcgaggat ggttctgtgc aactggcaga
     6901 tcattaccaa cagaatactc ctataggaga tgggccagtc ctcctgccag acaatcatta
     6961 cttgttcatc cagtccgcat tgtctaaaga tcctaacgag aaacgggacc atatggtatt
     7021 gctcgaattt gtcactgccg caggaataac acatggtatg gatgagctct ataagtaacg
     7081 tacgaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     7141 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     7201 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     7261 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     7321 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     7381 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     7441 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     7501 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     7561 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     7621 tctttgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgac
//
