LOCUS       sequence-361927-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a NT.2-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.2#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..81)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        253..632
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        634..832
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     787..807
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             850..1030
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1077..1202
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1699..1932
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2117..2161
                     /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    2459..2576
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2658..2906
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2658..2678
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2829..2848
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2927..3002
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3055..3554
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3090..3113)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3487..3506
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3558..3578
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3579..4607
                     /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    4663..5251
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4716..4736)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5134..5145)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5254..5270)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5255..5271)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5780..5960
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6022..6610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6102..6121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6781..7572,7573..7641))
                     /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     7404..7423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7642..7746)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
       61 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      121 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
      181 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      241 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      301 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      361 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      421 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      481 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      541 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      601 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      661 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      721 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      781 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
      841 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
      901 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
      961 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     1021 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     1081 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     1141 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     1201 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     1261 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     1321 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     1381 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     1441 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     1501 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag
     1561 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1621 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1681 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1741 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1801 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1861 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1921 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1981 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     2041 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     2101 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     2161 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     2221 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     2281 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     2341 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     2401 gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt
     2461 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2521 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2581 tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc tagccctgca
     2641 ggaactgttt aaactgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2701 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2761 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2821 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2881 atatcttgtg gaaaggacca aacaccgacg tagccttccg aaatatgttt tagagctaga
     2941 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     3001 gcttttttgt ttggcgcgcc tatggcttaa ttaacatggc ggccgcattc taccgggtag
     3061 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     3121 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     3181 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     3241 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     3301 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     3361 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     3421 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     3481 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     3541 ctccgggacc cgggatgccc aagaagaaga ggaaggtgtc caatttactg accgtacacc
     3601 aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg
     3661 acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt
     3721 gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg
     3781 aagatgttcg cgattatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc
     3841 agcaacattt gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg
     3901 acagcaatgc tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg
     3961 aacgtgcaaa acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca
     4021 tggaaaatag cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata
     4081 acaccctgtt acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg
     4141 acggtgggag aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg
     4201 tagagaaggc acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg
     4261 gtgtagctga tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg
     4321 cgccatctgc caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc
     4381 atcgattgat ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac
     4441 acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga
     4501 tcatgcaagc tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg
     4561 atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg cgattagcca tgtttaaggg
     4621 ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac
     4681 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4741 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4801 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4861 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     4921 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     4981 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5041 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5101 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5161 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5221 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg atcgagacct
     5281 agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg
     5341 gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag
     5401 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     5461 ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac
     5521 acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact
     5581 gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa
     5641 tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga
     5701 gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga
     5761 gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     5821 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     5881 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     5941 agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6001 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6061 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6121 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6181 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6241 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6301 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6361 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6421 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6481 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6541 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6601 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6661 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6721 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6781 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6841 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6901 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6961 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7021 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7081 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7141 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7201 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7261 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7321 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7381 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7441 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7501 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7561 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7621 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7681 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7741 tccgcgcaca tttccccga
//
