LOCUS       sequence-363973-        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-sgNT-hU6-sgLkb1_1st
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             71..251
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(313..901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(393..412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1072..1863,1864..1932))
                     /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     1695..1714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1933..2037)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2112..2131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2303..2682
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2684..2882
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2837..2857
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2900..3080
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3127..3252
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3749..3982
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4167..4211
                     /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    4509..4626
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     4939..4959
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5184..5204
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5355..5374
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5594..6093
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5629..5652)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6026..6045
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6097..6117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6118..7146
                     /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    7202..7790
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7255..7275)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7673..7684)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7793..7809)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7794..7810)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc
       61 ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      121 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      181 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      241 aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
      301 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
      361 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      421 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
      481 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
      541 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
      601 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      661 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      721 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
      781 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      841 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
      901 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
      961 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1021 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1081 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1141 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1201 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1261 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1321 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     1381 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     1441 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     1501 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     1561 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     1621 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     1681 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     1741 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     1801 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     1861 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     1921 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     1981 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2041 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     2101 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     2161 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     2221 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     2281 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     2341 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     2401 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     2461 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2521 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2581 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2641 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2701 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2761 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2821 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2881 agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     2941 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     3001 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     3061 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     3121 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     3181 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     3241 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     3301 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     3361 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     3421 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     3481 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     3541 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc
     3601 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     3661 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     3721 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     3781 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     3841 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     3901 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     3961 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     4021 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     4081 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     4141 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     4201 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     4261 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     4321 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     4381 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     4441 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     4501 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     4561 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     4621 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
     4681 tagccctgca ggaactgttt aaactgtgat ccgacgcgcc atctctaggc ccgcgccggc
     4741 cccctcgcac ggacttgtgg gagaagctcg gctactcccc tgccccggtt aatttgcata
     4801 taatatttcc tagtaactat agaggcttaa tgtgcgataa aagacagata atctgttctt
     4861 tttaatacta gctacatttt acatgatagg cttggatttc tataagagat acaaatacta
     4921 aattattatt ttaaaaaaca gcacaaaagg aaactcaccc taactgtaaa gtaattgtgt
     4981 gttttgagac tataagtatc ccttggagaa ccaccttgtt ggtggtgggc cgcagtcaca
     5041 agtttgagag ctaagcagaa agctgcatag caagttcaaa taaggctagt ccgtacacaa
     5101 cttgaaaaag tggcagccga gtcggctgct ttttttgctc gaatctacac tcagctatgg
     5161 cgcgccccaa ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata
     5221 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     5281 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     5341 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     5401 ctttatatat cttgtggaaa gccagaaaca tggcgaggta ttcggctccg cggtttcaga
     5461 gctaagcaca agagtgcata gcaagttgaa ataaggctag tccgtttaca acttgaaaaa
     5521 gtggcacccg agtcgggtgc tttttttgtt tggcgcgcct atggcttaat taacatggcg
     5581 gccgcattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     5641 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     5701 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     5761 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     5821 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     5881 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     5941 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     6001 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     6061 ctgttctcct cttcctcatc tccgggaccc gggatgccca agaagaagag gaaggtgtcc
     6121 aatttactga ccgtacacca aaatttgcct gcattaccgg tcgatgcaac gagtgatgag
     6181 gttcgcaaga acctgatgga catgttcagg gatcgccagg cgttttctga gcatacctgg
     6241 aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa
     6301 tggtttcccg cagaacctga agatgttcgc gattatcttc tatatcttca ggcgcgcggt
     6361 ctggcagtaa aaactatcca gcaacatttg ggccagctaa acatgcttca tcgtcggtcc
     6421 gggctgccac gaccaagtga cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa
     6481 gaaaacgttg atgccggtga acgtgcaaaa caggctctag cgttcgaacg cactgatttc
     6541 gaccaggttc gttcactcat ggaaaatagc gatcgctgcc aggatatacg taatctggca
     6601 tttctgggga ttgcttataa caccctgtta cgtatagccg aaattgccag gatcagggtt
     6661 aaagatatct cacgtactga cggtgggaga atgttaatcc atattggcag aacgaaaacg
     6721 ctggttagca ccgcaggtgt agagaaggca cttagcctgg gggtaactaa actggtcgag
     6781 cgatggattt ccgtctctgg tgtagctgat gatccgaata actacctgtt ttgccgggtc
     6841 agaaaaaatg gtgttgccgc gccatctgcc accagccagc tatcaactcg cgccctggaa
     6901 gggatttttg aagcaactca tcgattgatt tacggcgcta aggatgactc tggtcagaga
     6961 tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct
     7021 ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg
     7081 aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc
     7141 gattagccat gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga
     7201 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7261 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7321 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7381 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7441 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7501 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7561 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     7621 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     7681 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     7741 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     7801 tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     7861 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     7921 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     7981 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
     8041 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc
     8101 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag
     8161 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc
     8221 atgggatgga tgacccggag agagaagt
//
