LOCUS       sequence-361934-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Tet2-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-sgTet2#1/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..102)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        274..653
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        655..853
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     808..828
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             871..1051
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1098..1223
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1720..1953
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2138..2182
                     /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    2480..2597
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2679..2927
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2679..2699
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2850..2869
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2948..3023
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3076..3575
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3111..3134)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3508..3527
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3579..3599
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3600..4628
                     /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    4684..5272
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4737..4757)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5155..5166)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5275..5291)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5276..5292)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5801..5981
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6043..6631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6123..6142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6802..7593,7594..7662))
                     /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     7425..7444
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7663..7759,1..8))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
       61 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
      121 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
      181 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
      241 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
      301 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      361 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      421 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      481 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      541 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      601 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      661 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      721 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      781 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      841 tctatataag cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc
      901 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
      961 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     1021 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga
     1081 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa
     1141 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg
     1201 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa
     1261 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa
     1321 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta
     1381 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat
     1441 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca
     1501 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag
     1561 cggccggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
     1621 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
     1681 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
     1741 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
     1801 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
     1861 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
     1921 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
     1981 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
     2041 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
     2101 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
     2161 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
     2221 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
     2281 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
     2341 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag
     2401 agacagatcc attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa
     2461 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2521 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2581 caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt tggttagtac
     2641 cgggcccgct ctagccctgc aggaactgtt taaactgtga gggcctattt cccatgattc
     2701 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     2761 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     2821 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     2881 atttcgattt cttggcttta tatatcttgt ggaaaggacc aaacaccgga gaaattgatc
     2941 tgcgctggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     3001 aaaaagtggc accgagtcgg tgcttttttg tttggcgcgc ctatggctta attaacatgg
     3061 cggccgcatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     3121 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     3181 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     3241 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     3301 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     3361 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     3421 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     3481 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     3541 cgctgttctc ctcttcctca tctccgggac ccgggatgcc caagaagaag aggaaggtgt
     3601 ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg
     3661 aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct
     3721 ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga
     3781 aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg
     3841 gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt catcgtcggt
     3901 ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa
     3961 aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt
     4021 tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg
     4081 catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg
     4141 ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa
     4201 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg
     4261 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg
     4321 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg
     4381 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga
     4441 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg
     4501 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca
     4561 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg
     4621 gcgattagcc atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc
     4681 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4741 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4801 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4861 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4921 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4981 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     5041 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     5101 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     5161 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     5221 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     5281 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     5341 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     5401 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5461 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     5521 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     5581 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     5641 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     5701 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     5761 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     5821 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5881 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     5941 atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca
     6001 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6061 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6481 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6781 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6841 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6901 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6961 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7021 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7081 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7141 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7201 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7261 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7321 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7381 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7441 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7501 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7561 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7621 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7681 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7741 gaaaaataaa caaataggg
//
