LOCUS       sequence-361632-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a NT.3-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  AUTHORS   Cai H, Chew SK, Li C, Tsai MK, Andrejka L, Murray CW, Hughes NW, 
            Shuldiner EG, Ashkin EL, Tang R, Hung KL, Chen LC, Lee SYC, Yousefi 
            M, Lin WY, Kunder CA, Cong L, McFarland CD, Petrov DA, Swanton C, 
            Winslow MM
  TITLE     A functional taxonomy of tumor suppression in oncogenic KRAS-driven 
            lung cancer.
  JOURNAL   Cancer Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325.
  PUBMED    33608386
REFERENCE   2  (bases 1 to 7759)
  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: "Cancer 
            Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325."
COMMENT     Sequence Label: Lenti-sgNT.3#3/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(17..121)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(196..215)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        387..766
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        768..966
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     921..941
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             984..1164
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1211..1336
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1833..2066
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2251..2295
                     /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    2593..2710
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2792..3040
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2792..2812
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2963..2982
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3061..3136
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3189..3688
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3224..3247)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3621..3640
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3692..3712
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3713..4741
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    4797..5385
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4850..4870)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5268..5279)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5388..5404)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5389..5405)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5914..6094
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6156..6744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6236..6255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6915..7706,7707..7759,1..16))
                     /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     7538..7557
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
       61 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
      121 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
      181 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
      241 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
      301 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      361 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      421 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      481 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      541 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      601 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      661 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      721 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      781 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      841 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      901 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      961 aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
     1021 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     1081 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     1141 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     1201 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     1261 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     1321 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     1381 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     1441 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     1501 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     1561 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     1621 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg
     1681 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1741 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1801 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1861 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1921 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1981 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     2041 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     2101 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     2161 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     2221 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     2281 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     2341 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     2401 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     2461 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     2521 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt
     2581 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2641 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2701 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttag taccgggccc
     2761 gctctagccc tgcaggaact gtttaaactg tgagggccta tttcccatga ttccttcata
     2821 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     2881 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2941 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     3001 tttcttggct ttatatatct tgtggaaagg accaaacacc gactgaaatc caaggactgt
     3061 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     3121 ggcaccgagt cggtgctttt ttgtttggcg cgcctatggc ttaattaaca tggcggccgc
     3181 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     3241 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     3301 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     3361 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     3421 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     3481 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     3541 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
     3601 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     3661 ctcctcttcc tcatctccgg gacccgggat gcccaagaag aagaggaagg tgtccaattt
     3721 actgaccgta caccaaaatt tgcctgcatt accggtcgat gcaacgagtg atgaggttcg
     3781 caagaacctg atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat
     3841 gcttctgtcc gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt
     3901 tcccgcagaa cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc
     3961 agtaaaaact atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct
     4021 gccacgacca agtgacagca atgctgtttc actggttatg cggcggatcc gaaaagaaaa
     4081 cgttgatgcc ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca
     4141 ggttcgttca ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct
     4201 ggggattgct tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga
     4261 tatctcacgt actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt
     4321 tagcaccgca ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg
     4381 gatttccgtc tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa
     4441 aaatggtgtt gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat
     4501 ttttgaagca actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct
     4561 ggcctggtct ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt
     4621 ttcaataccg gagatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta
     4681 tatccgtaac ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgatta
     4741 gccatgttta agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc
     4801 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     4861 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     4921 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     4981 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     5041 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     5101 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     5161 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     5221 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     5281 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     5341 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac
     5401 ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat
     5461 gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct
     5521 caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     5581 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg
     5641 tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg
     5701 atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag
     5761 gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg
     5821 atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat
     5881 cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga
     5941 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     6001 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     6061 agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag
     6121 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6181 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6241 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6301 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6361 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6421 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6481 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6541 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6601 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6661 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6721 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6781 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6841 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6901 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6961 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7021 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7081 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7141 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7201 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7261 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7321 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7381 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7441 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7501 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7561 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7621 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7681 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7741 ggaataaggg cgacacgga
//
