LOCUS       sequence-484989-        7996 bp    DNA     circular SYN 28-AUG-2025
DEFINITION  For lentiviral Crispr/Cas9 mediated knockout of mouse RXRa, using a 
            guide RNA against exon 4, coupled to EF1a-driven mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7996)
  AUTHORS   Stewart KS, Abdusselamoglu MD, Tierney MT, Gola A, Hur YH, Gonzales 
            KAU, Yuan S, Bonny AR, Yang Y, Infarinato NR, Cowley CJ, Levorse JM,
            Pasolli HA, Ghosh S, Rothlin CV, Fuchs E
  TITLE     Stem cells tightly regulate dead cell clearance to maintain tissue 
            fitness.
  JOURNAL   Nature. 2024 Sep;633(8029):407-416. doi: 10.1038/s41586-024-07855-6.
            Epub 2024 Aug 21.
  PUBMED    39169186
REFERENCE   2  (bases 1 to 7996)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-024-07855-6"; journalName: "Nature"; date: 
            "2024-09"; volume: "633"; issue: "8029"; pages: "407-416"
COMMENT     Sequence Label: pLKO-U6-sgRXRa4-1; EF1a-mScarlet
FEATURES             Location/Qualifiers
     source          1..7996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(9..437)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(119..140)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     331..350
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     564..586
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     578..595
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     579..595
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     605..624
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        605..623
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      644..779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    819..940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(830..849)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     884..903
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             1012..1245
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(1362..2057)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1637..1656)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1906..1924
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2054..2063
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2086..3264)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2095..3033
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2118..2138)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    3403..3520
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(3582..3657)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3679..3927)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3737..3756)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3907..3927)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(4225..4269)
                     /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    4454..4687
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5180..5305
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             5352..5532
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5533..5759)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(5787..5807)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5787..5805)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5826..5842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5826..5842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5839..5861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5850..5866
                     /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(5874..5880,5881..5898,5899..5904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5919..5940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6057..6074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6228..6816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6308..6327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6987..7778,7779..7847))
                     /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     7610..7629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7848..7952)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
       61 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
      121 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
      181 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
      241 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
      301 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
      361 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
      421 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
      481 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      541 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
      601 cgcgtaatac gactcactat agggcgaatt gggtacgtcc ctaggcctcc aaaaaagcct
      661 cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa
      721 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg
      781 ggcggagtta ggggcgggat agctagagcc agacatgata agatacattg atgagtttgg
      841 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
      901 tgctttattt gtaaccatta taagctgcaa taaacaagtt cctctcactc tctgatattc
      961 atttctttgc aagttataaa tactgaataa taagatgaca tgaactacta ctgctagaga
     1021 ttttccacac tgactaaaag ggtctgaggg atctctagtt accagagtca cacaacagac
     1081 gggcacacac tacttgaagc actcaaggca agctttattg aggcttaagc agtgggttcc
     1141 ctagttagcc agagagctcc caggctcaga tctggtctaa ccagagagac ccagtacaag
     1201 caaaaagcag atcttgtctt cgttgggagt gaattagccc ttccagtccc cccttttctt
     1261 ttaaaaagtg gctaagatct acagctgcct tgtaagtcat tggtcttaaa ggtaccgtcg
     1321 actgcagaat tcgaagcttg agctcgagat ctgagtccgg acttgtacag ctcgtccatg
     1381 ccgccggtgg agtggcggcc ctcggagcgt tcgtactgtt ccaccacggt gtagtcctcg
     1441 ttgtgggagg tgatgtccaa cttgcggtcg acgttgtagg cgccgggcat ctgcacgggc
     1501 ttcttggcct tgtaggtggt cttgaagtcc gccaggtagc ggccgccgtc cttcaggcgc
     1561 agggccatct taatgtcgcc cttcagcacg ccgtcctcgg ggtacaaccg ctcggtggac
     1621 gcttcccagc ccattgtctt cttctgcatt acggggccgt caggagggaa gttggtgccg
     1681 cggagcttca ccttgtagat cagggtgccg tcctccaggg aggtgtcctg ggtcacggtc
     1741 acggcgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac
     1801 tgcttatagt agtcggggat gtcggcgggg tgcttggtga aggccctgga gccgtacatg
     1861 aactgagggg acaggatgtc ccaggagaag ggcagggggc cacccttggt caccttcagc
     1921 ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg
     1981 tggccgttca tggagccctc catgtgcacc ttgaaccgca tgaactcctt gatcactgcc
     2041 tcgcccttgc tcaccatggt ggcgaccggt agcgctagcg gatcctcacg acacctgaaa
     2101 tggaagaaaa aaactttgaa ccactgtctg aggcttgaga atgaaccaag atccaaactc
     2161 aaaaagggca aattccaagg agaattacat caagtgccaa gctggcctaa cttcagtctc
     2221 cacccactca gtgtggggaa actccatcgc ataaaacccc tccccccaac ctaaagacga
     2281 cgtactccaa aagctcgaga actaatcgag gtgcctggac ggcgcccggt actccgtgga
     2341 gtcacatgaa gcgacggctg aggacggaaa ggcccttttc ctttgtgtgg gtgactcacc
     2401 cgcccgctct cccgagcgcc gcgtcctcca ttttgagctc cctgcagcag ggccgggaag
     2461 cggccatctt tccgctcacg caactggtgc cgaccgggcc agccttgccg cccagggcgg
     2521 ggcgatacac ggcggcgcga ggccaggcac cagagcaggc cggccagctt gagactaccc
     2581 ccgtccgatt ctcggtggcc gcgctcgcag gccccgcctc gccgaacatg tgcgctggga
     2641 cgcacgggcc ccgtcgccgc ccgcggcccc aaaaaccgaa ataccagtgt gcagatcttg
     2701 gcccgcattt acaagactat cttgccagaa aaaaagcgtc gcagcaggtc atcaaaaatt
     2761 ttaaatggct agagacttat cgaaagcagc gagacaggcg cgaaggtgcc accagattcg
     2821 cacgcggcgg ccccagcgcc caggccaggc ctcaactcaa gcacgaggcg aaggggctcc
     2881 ttaagcgcaa ggcctcgaac tctcccaccc acttccaacc cgaagctcgg gatcaagaat
     2941 cacgtactgc agccaggtgg aagtaattca aggcacgcaa gggccataac ccgtaaagag
     3001 gccaggcccg cgggaaccac acacggcact tacctgtgtt ctggcggcaa acccgttgcg
     3061 aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa
     3121 aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc
     3181 accggatcaa ttgccgaccc ctccccccaa cttctcgggg actgtgggcg atgtgcgctc
     3241 tgcccactga cgggcaccgg agccaattcc cactcctttc aagacctaga aggtccatta
     3301 gctgcaaaga ttcctctctg tttaaaactt tatccatctt tgcacccggg ccccctcgag
     3361 ccggcgccaa agtggatctc tgctgtccct gtaataaacc cgaaaatttt gaatttttgt
     3421 aatttgtttt tgtaattctt tagtttgtat gtctgttgct attatgtcta ctattctttc
     3481 ccctgcactg taccccccaa tccccccttt tcttttaaaa ttgtggatga atactgccat
     3541 ttgtctcaag atctagttac gccaagcttg aattcaaaaa agcaccgact cggtgccact
     3601 ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct ctaaaacgcc
     3661 gagacaacaa ggactgccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
     3721 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
     3781 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
     3841 gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta tatgcaaata
     3901 tgaaggaatc atgggaaata ggccctcggt gaagggggcg gccgctcgag gctagtctcg
     3961 tgatcgatac cgtcgagatc cgttcactaa tcgaatggat ctgtctctgt ctctctctcc
     4021 accttcttct tctattcctt cgggcctgtc gggtcccctc ggggttggga ggtgggtctg
     4081 aaacgataat ggtgaatatc cctgcctaac tctattcact atagaaagta cagcaaaaac
     4141 tattcttaaa cctaccaagc ctcctactat cattatgaat aattttatat accacagcca
     4201 atttgttatg ttaaaccaat tccacaaact tgcccattta tctaattcca ataattcttg
     4261 ttcattcttt tcttgctggt tttgcgattc ttcaattaag gagtgtatta agcttgtgta
     4321 attgttaatt tctctgtccc actccatcca ggtcgtgtga ttccaaatct gttccagaga
     4381 tttattactc caactagcat tccaaggcac agcagtggtg caaatgagtt ttccagagca
     4441 accccaaatc cccaggagct gttgatcctt taggtatctt tccacagcca ggattcttgc
     4501 ctggagctgc ttgatgcccc agactgtgag ttgcaacaga tgctgttgcg cctcaatagc
     4561 cctcagcaaa ttgttctgct gctgcactat accagacaat aattgtctgg cctgtaccgt
     4621 cagcgtcatt gacgctgcgc ccatagtgct tcctgctgct cccaagaacc caaggaacaa
     4681 agctcctatt cccactgctc ttttttctct ctgcaccact cttctctttg ccttggtggg
     4741 tgctactcct aatggttcaa tttttactac tttatattta tataattcac ttctccaatt
     4801 gtccctcata tctcctcctc caggtctgaa gatcagcggc cgcttgctgt gcggtggtct
     4861 tacttttgtt ttgctcttcc tctatcttgt ctaaagcttc cttggtgtct tttatctcta
     4921 tcctttgatg cacacaatag agggttgcta ctgtattata taatgatcta agttcttctg
     4981 atcctgtctg aagggatggt tgtagctgtc ccagtatttg tctacagcct tctgatgttt
     5041 ctaacaggcc aggattaact gcgaatcgtt ctagctccct gcttgcccat actatatgtt
     5101 ttaatttata ttttttcttt ccccctggcc ttaaccgaat tttttcccat cgcgatctaa
     5161 ttctcccccg cttaatactg acgctctcgc acccatctct ctccttctag cctccgctag
     5221 tcaaaatttt tggcgtactc accagtcgcc gcccctcgcc tcttgccgtg cgcgcttcag
     5281 caagccgagt cctgcgtcga gagagctcct ctggtttccc tttcgctttc aagtccctgt
     5341 tcgggcgcca ctgctagaga ttttccacac tgactaaaag ggtctgaggg atctctagtt
     5401 accagagtca cacaacagac gggcacacac tacttgaagc actcaaggca agctttattg
     5461 aggcttaagc agtgggttcc ctagttagcc agagagctcc caggctcaga tctggtctaa
     5521 ccagagagac ccgtttatgt atcgagctag gcacttaaat acaatatctc tgcaatgcgg
     5581 caattcagtg gttcgtccaa tccatgtcag acccgtctgt tgccttccta ataaggcacg
     5641 atcgtaccac cttacttcca ccaatcggca tgcacggtgc tttttctctc cttgtaaggc
     5701 atgttgctaa ctcatcgtta ccatgttgca agactacaag agtattgcat aagactacat
     5761 taagcttgca gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     5821 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5881 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6001 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6061 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6121 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6181 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6241 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6301 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6361 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6421 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6481 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6541 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6601 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6661 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6721 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6781 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6841 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6901 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6961 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7021 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7081 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7141 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7201 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7261 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7321 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7381 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7441 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7501 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7561 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7621 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7681 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7741 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7801 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7861 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7921 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     7981 ttgtaagcgt taatat
//
