LOCUS       sequence-350037-        8248 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 8248)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8248)
  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_1st-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             109..289
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(351..939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(431..450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1110..1901,1902..1970))
                     /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     1733..1752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1971..2075)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2150..2169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2341..2720
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2722..2920
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2875..2895
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2938..3118
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3165..3290
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3787..4020
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4205..4249
                     /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    4547..4664
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     4977..4997
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5222..5242
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5393..5412
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5632..6131
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5667..5690)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6064..6083
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6135..6155
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6156..7184
                     /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    7240..7828
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7293..7313)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7711..7722)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7831..7847)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7832..7848)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
       61 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
      121 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      181 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      241 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
      301 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      361 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      421 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      481 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      541 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      601 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      661 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      721 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      781 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      841 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      901 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      961 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1021 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1081 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1141 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1201 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1261 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1321 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     1381 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     1441 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1501 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1561 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1621 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1681 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1741 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1801 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1861 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1921 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1981 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2041 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     2101 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     2161 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     2221 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     2281 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     2341 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     2401 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     2461 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     2521 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2581 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2641 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2701 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2761 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2821 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2881 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
     2941 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     3001 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     3061 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     3121 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     3181 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     3241 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     3301 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     3361 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     3421 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     3481 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     3541 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     3601 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     3661 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     3721 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     3781 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     3841 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     3901 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     3961 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     4021 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     4081 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     4141 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     4201 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     4261 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     4321 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     4381 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     4441 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     4501 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     4561 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     4621 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     4681 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gccctgcagg aactgtttaa
     4741 actgtgatcc gacgcgccat ctctaggccc gcgccggccc cctcgcacgg acttgtggga
     4801 gaagctcggc tactcccctg ccccggttaa tttgcatata atatttccta gtaactatag
     4861 aggcttaatg tgcgataaaa gacagataat ctgttctttt taatactagc tacattttac
     4921 atgataggct tggatttcta taagagatac aaatactaaa ttattatttt aaaaaacagc
     4981 acaaaaggaa actcacccta actgtaaagt aattgtgtgt tttgagacta taagtatccc
     5041 ttggagaacc accttgttgg cgaggtattc ggctccgcgg tttgagagct aagcagaaag
     5101 ctgcatagca agttcaaata aggctagtcc gtacacaact tgaaaaagtg gcagccgagt
     5161 cggctgcttt ttttgctcga atctacactc agctatggcg cgccccaagg tcgggcagga
     5221 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     5281 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     5341 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     5401 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagc
     5461 cagaaacatg gtggtgggcc gcagtcacaa gtttcagagc taagcacaag agtgcatagc
     5521 aagttgaaat aaggctagtc cgtttacaac ttgaaaaagt ggcacccgag tcgggtgctt
     5581 tttttgtttg gcgcgcctat ggcttaatta acatggcggc cgcattctac cgggtagggg
     5641 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     5701 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     5761 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     5821 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     5881 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     5941 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     6001 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     6061 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     6121 cgggacccgg gatgcccaag aagaagagga aggtgtccaa tttactgacc gtacaccaaa
     6181 atttgcctgc attaccggtc gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca
     6241 tgttcaggga tcgccaggcg ttttctgagc atacctggaa aatgcttctg tccgtttgcc
     6301 ggtcgtgggc ggcatggtgc aagttgaata accggaaatg gtttcccgca gaacctgaag
     6361 atgttcgcga ttatcttcta tatcttcagg cgcgcggtct ggcagtaaaa actatccagc
     6421 aacatttggg ccagctaaac atgcttcatc gtcggtccgg gctgccacga ccaagtgaca
     6481 gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga aaacgttgat gccggtgaac
     6541 gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg
     6601 aaaatagcga tcgctgccag gatatacgta atctggcatt tctggggatt gcttataaca
     6661 ccctgttacg tatagccgaa attgccagga tcagggttaa agatatctca cgtactgacg
     6721 gtgggagaat gttaatccat attggcagaa cgaaaacgct ggttagcacc gcaggtgtag
     6781 agaaggcact tagcctgggg gtaactaaac tggtcgagcg atggatttcc gtctctggtg
     6841 tagctgatga tccgaataac tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc
     6901 catctgccac cagccagcta tcaactcgcg ccctggaagg gatttttgaa gcaactcatc
     6961 gattgattta cggcgctaag gatgactctg gtcagagata cctggcctgg tctggacaca
     7021 gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg agtttcaata ccggagatca
     7081 tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa ctatatccgt aacctggata
     7141 gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga ttagccatgt ttaagggttc
     7201 cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     7261 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     7321 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     7381 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     7441 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     7501 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     7561 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     7621 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     7681 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     7741 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     7801 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga
     7861 aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct
     7921 agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc
     7981 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     8041 agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca
     8101 aggctacttc cctgattggc agaactacac accagggcca gggatcagat atccactgac
     8161 ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga
     8221 aggagagaac acccgcttgt tacaccct
//
