LOCUS       sequence-363649-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Kdm6a-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 7760)
  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-sgKdm6a#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(156..175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        347..726
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        728..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     881..901
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             944..1124
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1171..1296
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1793..2026
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2211..2255
                     /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    2553..2670
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2752..3000
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2752..2772
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2923..2942
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3022..3097
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3150..3649
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3185..3208)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3582..3601
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3653..3673
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3674..4702
                     /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    4758..5346
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4811..4831)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5229..5240)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5349..5365)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5350..5366)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5875..6055
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6117..6705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6197..6216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6876..7667,7668..7736))
                     /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     7499..7518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7737..7760,1..81))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
       61 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
      121 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
      181 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
      241 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
      301 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      361 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      421 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      481 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      541 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      601 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      661 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      721 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      781 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      841 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      901 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
      961 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     1021 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     1081 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     1141 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     1201 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     1261 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     1321 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     1381 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     1441 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1501 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1561 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1621 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     1681 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1741 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1801 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1861 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1921 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1981 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     2041 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     2101 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     2161 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     2221 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     2281 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     2341 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     2401 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     2461 gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa
     2521 ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2581 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2641 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2701 gtttggttag taccgggccc gctctagccc tgcaggaact gtttaaactg tgagggccta
     2761 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     2821 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     2881 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     2941 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg accaaacacc
     3001 gaggttggat gcatcacact ggttttagag ctagaaatag caagttaaaa taaggctagt
     3061 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgtttggc gcgcctatgg
     3121 cttaattaac atggcggccg cattctaccg ggtaggggag gcgcttttcc caaggcagtc
     3181 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
     3241 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
     3301 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
     3361 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
     3421 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
     3481 cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggngggct
     3541 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
     3601 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggacccggga tgcccaagaa
     3661 gaagaggaag gtgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga
     3721 tgcaacgagt gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt
     3781 ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa
     3841 gttgaataac cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata
     3901 tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat
     3961 gcttcatcgt cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat
     4021 gcggcggatc cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt
     4081 cgaacgcact gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga
     4141 tatacgtaat ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat
     4201 tgccaggatc agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat
     4261 tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt
     4321 aactaaactg gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta
     4381 cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc
     4441 aactcgcgcc ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga
     4501 tgactctggt cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg
     4561 agatatggcc cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa
     4621 tgtaaatatt gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg
     4681 cctgctggaa gatggcgatt agccatgttt aagggttccg gttccactag gtacaattcg
     4741 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     4801 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     4861 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     4921 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     4981 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5041 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5101 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     5161 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     5221 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     5281 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     5341 ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc aatcacaagt
     5401 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
     5461 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
     5521 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     5581 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
     5641 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
     5701 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
     5761 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
     5821 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
     5881 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     5941 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     6001 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagcatg
     6061 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6121 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6181 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6241 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6301 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6361 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6421 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6481 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6541 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6601 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6661 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6721 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6781 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6841 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6901 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6961 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7021 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7081 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7141 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7201 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7261 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7321 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7381 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7441 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7501 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7561 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7621 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7681 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7741 ttcctttttc aatattattg
//
