LOCUS       sequence-337087-        8120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To knockout murine XAF1 gene by CRISPR-Cas9 system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8120)
  TITLE     murine XAF1 knockout
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8120)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKLV-XAF1
FEATURES             Location/Qualifiers
     source          1..8120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     233..252
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             340..936
                     /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(340..359)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     836..856
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             955..1008
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1009..1710
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(2065..2082)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2071..2295
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2321..2341)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2321..2339)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2360..2376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2360..2376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2373..2395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2384..2400
                     /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(2408..2414,2415..2432,2433..2438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2453..2474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2591..2608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2762..3350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2842..2861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3521..4312,4313..4381))
                     /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     4144..4163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4382..4486)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4512..4967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4649..4670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4861..4880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5094..5116
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5108..5125
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5109..5125
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5135..5154
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5135..5153
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        5224..5527
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5529..5727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5682..5702
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5745..5925
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5972..6097
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6594..6827
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7012..7056
                     /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    7354..7471
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7554..7802
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7554..7574
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7725..7744
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7823..7898
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        join(7921..8120,1..300)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7956..7979)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
       61 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
      121 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
      181 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
      241 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
      301 acctgcatcc atctagatct cgagcagctg aagcttacca tgaccgagta caagcccacg
      361 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
      421 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
      481 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
      541 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
      601 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
      661 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
      721 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
      781 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
      841 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
      901 ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggcg gcgggtccgg aggagagggc
      961 agaggaagtc tcctaacatg cggtgacgtg gaggagaatc ctggcccaat gagcgagctg
     1021 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
     1081 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     1141 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
     1201 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     1261 cctgagggct tcacatggga gagagtcacc acatacgagg acgggggcgt gctgaccgct
     1321 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     1381 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     1441 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     1501 gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct
     1561 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     1621 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     1681 cctagcaaac tggggcacaa gcttaattga gcggccgcta ggtaccttta agaccaatga
     1741 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     1801 taattcactc ccaaagaagt caagatctgc tttttgcctg tactgggtct ctctggttag
     1861 accagagtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     1921 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     1981 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     2041 tagcagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2101 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2161 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2221 gggtggggca ggacagcaag ggggaggatt gggaagtcaa tagcaggcat gctggggatg
     2281 cggtgggctc tatgggcggc cgttaattaa gcttttgttc cctttagtga gggttaattg
     2341 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2401 ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga
     2461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3181 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4381 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4441 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4501 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4561 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4621 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4681 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4741 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4801 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4861 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4921 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     4981 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     5041 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     5101 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
     5161 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     5221 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     5281 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     5341 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     5401 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     5461 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     5521 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     5581 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     5641 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     5701 tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag
     5761 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5821 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5881 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     5941 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     6001 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     6061 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     6121 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     6181 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     6241 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     6301 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     6361 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     6421 caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     6481 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     6541 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     6601 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     6661 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     6721 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     6781 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     6841 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     6901 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     6961 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     7021 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     7081 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     7141 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     7201 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     7261 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     7321 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     7381 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     7441 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     7501 agtttggtta gtaccgggcc ctacgcgtta ctcgagccaa ggtcgggcag gaagagggcc
     7561 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     7621 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     7681 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     7741 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     7801 ccgggctgac ttccaagtgt gcgttttaga gctagaaata gcaagttaaa ataaggctag
     7861 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttggatc caattctacc
     7921 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     7981 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     8041 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     8101 ggaagttccc ccccgccccg
//
