LOCUS       sequence-350113-        8249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Lkb1-targeting and a non-targeting gRNAs and 
            Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8249)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8249)
  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"
COMMENT     Sequence Label: mU6-sgLkb1_2nd-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..94
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             146..166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             167..1195
                     /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    1251..1839
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1304..1324)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1722..1733)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1842..1858)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1843..1859)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2368..2548
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2610..3198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2690..2709)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3369..4160,4161..4229))
                     /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     3992..4011
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4230..4334)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4409..4428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4600..4979
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4981..5179
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5134..5154
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5197..5377
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5424..5549
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6046..6279
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6464..6508
                     /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    6806..6923
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     7236..7256
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     7481..7501
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7652..7671
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        join(7892..8249,1..142)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7927..7950)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc
       61 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc
      121 ttcctcatct ccgggacccg ggatgcccaa gaagaagagg aaggtgtcca atttactgac
      181 cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa
      241 cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct
      301 gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc
      361 agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa
      421 aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg
      481 accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga
      541 tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg
      601 ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat
      661 tgcttataac accctgttac gtatagccga aattgccagg atcagggtta aagatatctc
      721 acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac
      781 cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc
      841 cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg
      901 tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga
      961 agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg
     1021 gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat
     1081 accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg
     1141 taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg attagccatg
     1201 tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc
     1261 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     1321 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     1381 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     1441 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     1501 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     1561 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     1621 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     1681 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     1741 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     1801 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat
     1861 cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat
     1921 tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta
     1981 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     2041 gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc
     2101 taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga
     2161 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa
     2221 gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat
     2281 gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg
     2341 gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg
     2401 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     2461 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     2521 ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg
     2581 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2641 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2701 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2761 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2821 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2881 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2941 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3001 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3061 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3121 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3181 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3241 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3301 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3361 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3421 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3481 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3541 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3601 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3661 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3721 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3781 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3841 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3901 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3961 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4021 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4081 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4141 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4201 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4261 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4321 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     4381 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4441 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     4501 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     4561 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     4621 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     4681 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     4741 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     4801 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     4861 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     4921 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     4981 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     5041 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     5101 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     5161 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
     5221 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5281 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     5341 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
     5401 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
     5461 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
     5521 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
     5581 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     5641 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     5701 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     5761 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     5821 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     5881 agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     5941 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     6001 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     6061 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     6121 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     6181 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     6241 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     6301 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     6361 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     6421 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     6481 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     6541 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     6601 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     6661 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     6721 agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca
     6781 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     6841 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     6901 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt
     6961 tagtaccggg cccgctctag ccctgcagga actgtttaaa ctgtgatccg acgcgccatc
     7021 tctaggcccg cgccggcccc ctcgcacgga cttgtgggag aagctcggct actcccctgc
     7081 cccggttaat ttgcatataa tatttcctag taactataga ggcttaatgt gcgataaaag
     7141 acagataatc tgttcttttt aatactagct acattttaca tgataggctt ggatttctat
     7201 aagagataca aatactaaat tattatttta aaaaacagca caaaaggaaa ctcaccctaa
     7261 ctgtaaagta attgtgtgtt ttgagactat aagtatccct tggagaacca ccttgttggc
     7321 gaggtattcg gctccgcggt ttgagagcta agcagaaagc tgcatagcaa gttcaaataa
     7381 ggctagtccg tacacaactt gaaaaagtgg cagccgagtc ggctgctttt tttgctcgaa
     7441 tctacactca gctatggcgc gccccaaggt cgggcaggaa gagggcctat ttcccatgat
     7501 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     7561 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     7621 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     7681 gtatttcgat ttcttggctt tatatatctt gtggaaagcc agaaacatgg aagctgcgca
     7741 ggatccccaa gtttcagagc taagcacaag agtgcatagc aagttgaaat aaggctagtc
     7801 cgtttacaac ttgaaaaagt ggcacccgag tcgggtgctt tttttgtttg gcgcgcctat
     7861 ggcttaatta acatggcggc cgcattctac cgggtagggg aggcgctttt cccaaggcag
     7921 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     7981 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     8041 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     8101 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     8161 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     8221 ttcgctttct gggctcagag gctgggaag
//
