LOCUS       sequence-313413-        7553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lenti-viral construct with Cre recombinase and U6 driven sgRNA 
            against mouse Ripk4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7553)
  AUTHORS   Loganathan SK, Schleicher K, Malik A, Quevedo R, Langille E, Teng K,
            Oh RH, Rathod B, Tsai R, Samavarchi-Tehrani P, Pugh TJ, Gingras AC, 
            Schramek D
  TITLE     Rare driver mutations in head and neck squamous cell carcinomas 
            converge on NOTCH signaling.
  JOURNAL   Science. 2020 Mar 13;367(6483):1264-1269. doi: 
            10.1126/science.aax0902.
  PUBMED    32165588
REFERENCE   2  (bases 1 to 7553)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2020-03-13- 13"; volume: "367"; 
            issue: "6483"; pages: "1264-1269"
COMMENT     Sequence Label: pLKO-Cre sgRipk4 v3
FEATURES             Location/Qualifiers
     source          1..7553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      535..544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             547..567
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     LTR             1677..1910
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1982..2103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2019..2038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2073..2092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2143..2278
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2298..2317)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2299..2317)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2327..2344)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2327..2343)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2336..2358)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2485..2940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2572..2591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2782..2803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2966..3070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3071..3139,3140..3931)
                     /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(3289..3308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4102..4690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4591..4610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4844..4861
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4978..4999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5014..5019,5020..5037,5038..5044)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5052..5068
                     /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     5057..5079
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5076..5092
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5076..5092
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5111..5131
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5113..5131
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5159..5385
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5386..5566
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5613..5738
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6231..6464
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6649..6693
                     /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        6991..7239
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6991..7011
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7162..7181
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7261..7336
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    7384..7501
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7550..7553,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcaggtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatcccgggt cgacatcaag cttggccacc
      541 atggtgccca agaagaagag gaaagtctcc aacctgctga ctgtgcacca aaacctgcct
      601 gccctccctg tggatgccac ctctgatgaa gtcaggaaga acctgatgga catgttcagg
      661 gacaggcagg ccttctctga acacacctgg aagatgctcc tgtctgtgtg cagatcctgg
      721 gctgcctggt gcaagctgaa caacaggaaa tggttccctg ctgaacctga ggatgtgagg
      781 gactacctcc tgtacctgca agccagaggc ctggctgtga agaccatcca acagcacctg
      841 ggccagctca acatgctgca caggagatct ggcctgcctc gcccttctga ctccaatgct
      901 gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg atgctgggga gagagccaag
      961 caggccctgg cctttgaacg cactgacttt gaccaagtca gatccctgat ggagaactct
     1021 gacagatgcc aggacatcag gaacctggcc ttcctgggca ttgcctacaa caccctgctg
     1081 cgcattgccg aaattgccag aatcagagtg aaggacatct cccgcaccga tggtgggaga
     1141 atgctgatcc acattggcag gaccaagacc ctggtgtcca cagctggtgt ggagaaggcc
     1201 ctgtccctgg gggttaccaa gctggtggag agatggatct ctgtgtctgg tgtggctgat
     1261 gaccccaaca actacctgtt ctgccgggtc agaaagaatg gtgtggctgc cccttctgcc
     1321 acctcccaac tgtccacccg ggccctggaa gggatctttg aggccaccca ccgcctgatc
     1381 tatggtgcca aggatgactc tgggcagaga tacctggcct ggtctggcca ctctgccaga
     1441 gtgggtgctg ccagggacat ggccagggct ggtgtgtcca tccctgaaat catgcaggct
     1501 ggtggctgga ccaatgtgaa cattgtgatg aactacatca gaaacctgga ctctgagact
     1561 ggggccatgg tgaggcttct agaggatggg gactgaaaat gcatcggtac ctttaagacc
     1621 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     1681 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     1741 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     1801 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     1861 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     1921 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     1981 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2041 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2101 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     2161 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2221 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2281 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     2341 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2401 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2461 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2521 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2581 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2641 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2701 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2761 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2821 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2881 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2941 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3001 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3061 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3121 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3181 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3241 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3301 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3361 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3421 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3481 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3541 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3601 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3661 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3721 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3781 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3841 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3901 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3961 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4021 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4081 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4141 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4201 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4261 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4321 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4381 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4441 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4501 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4561 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4621 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4681 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4741 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4801 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4861 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4921 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     4981 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5041 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5101 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
     5161 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     5221 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     5281 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     5341 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     5401 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5461 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5521 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     5581 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     5641 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     5701 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     5761 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     5821 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     5881 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     5941 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     6001 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     6061 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     6121 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     6181 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     6241 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     6301 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     6361 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     6421 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     6481 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     6541 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     6601 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     6661 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     6721 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     6781 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     6841 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     6901 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga
     6961 gactagcctc gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat
     7021 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     7081 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     7141 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     7201 ttcttggctt tatatatctt gtggaaagga cgaaacaccg cgtgcacgat gcgaaagcgc
     7261 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     7321 ggcaccgagt cggtgctttt ttgaattctc gacctcgaga caaatggcag tattcatcca
     7381 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     7441 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     7501 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg ggg
//
