LOCUS       sequence-352659-        8673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neomycin(bU6), non-targeting (mU6) and Lkb1 (hU6) gRNAs 
            and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8673)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8673)
  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: bU6-sgNeo1-mU6-sgNT-hU6-sgLkb1_2nd
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(474..1062)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(554..573)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1233..2024,2025..2093))
                     /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     1856..1875
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2094..2198)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2273..2292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2464..2843
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2845..3043
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2998..3018
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3061..3241
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3288..3413
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3910..4143
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4328..4372
                     /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    4670..4787
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5524..5544
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5769..5789
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5940..5959
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6180..6679
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6215..6238)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6612..6631
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6683..6703
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6704..7732
                     /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    7788..8376
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7841..7861)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8259..8270)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8379..8395)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8380..8396)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta
       61 ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac
      121 cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac
      181 agccgcctag catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga
      421 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      481 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      541 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      601 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      661 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      721 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      781 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      841 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      901 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      961 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1021 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1081 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1141 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1201 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1261 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1321 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1381 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1441 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1501 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1561 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1621 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1681 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1741 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1801 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1861 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1921 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1981 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2041 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2101 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2161 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2221 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     2281 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     2341 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2401 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2461 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2521 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2581 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2641 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2701 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2761 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2821 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2881 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2941 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     3001 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact
     3061 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     3121 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     3181 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     3241 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
     3301 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
     3361 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
     3421 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
     3481 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     3541 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     3601 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     3661 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     3721 aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg
     3781 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     3841 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     3901 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     3961 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     4021 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     4081 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     4141 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     4201 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     4261 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     4321 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     4381 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     4441 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     4501 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
     4561 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
     4621 ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa
     4681 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     4741 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     4801 gggacagcag agatccagtt tggttagtac cgggcccgct ctagccctgc aggaactgtt
     4861 taaactgtcg tgaccgagct tgtctgcccg cgcagtccac tagacagacg cgcagaggtc
     4921 gggcgggcct agggccaatt tcccatggtt cccttcattt gcatatatga tgtaataagg
     4981 ttatggagac tattaaactt agccctaatc aaactatatg atgataatgt gtgtggtaca
     5041 aaaggtcata acttattata tactttgaaa cttaaaaaag ggttacagtt tagtcaccat
     5101 aactgtaaaa ttttttctat tcttagcttt atatagttct tgagaggcca tgtttatggt
     5161 catggctgat gcaatgcggg tttaagagct aagctggaaa cagcatagca agtttaaata
     5221 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttccagtctg
     5281 actatcacga ttgatccgac gcgccatctc taggcccgcg ccggccccct cgcacggact
     5341 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta
     5401 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac
     5461 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa
     5521 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa
     5581 gtatcccttg gagaaccacc ttgttggcga ggtattcggc tccgcggttt gagagctaag
     5641 cagaaagctg catagcaagt tcaaataagg ctagtccgta cacaacttga aaaagtggca
     5701 gccgagtcgg ctgctttttt tgctcgaatc tacactcagc tatggcgcgc cccaaggtcg
     5761 ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     5821 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     5881 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     5941 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     6001 ggaaagccag aaacatggaa gctgcgcagg atccccaagt ttcagagcta agcacaagag
     6061 tgcatagcaa gttgaaataa ggctagtccg tttacaactt gaaaaagtgg cacccgagtc
     6121 gggtgctttt tttgtttggc gcgcctatgg cttaattaac atggcggccg cattctaccg
     6181 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     6241 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     6301 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     6361 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     6421 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     6481 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     6541 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     6601 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     6661 ctcatctccg ggacccggga tgcccaagaa gaagaggaag gtgtccaatt tactgaccgt
     6721 acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc gcaagaacct
     6781 gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc
     6841 cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt ttcccgcaga
     6901 acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg cagtaaaaac
     6961 tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc tgccacgacc
     7021 aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa acgttgatgc
     7081 cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc aggttcgttc
     7141 actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc tggggattgc
     7201 ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag atatctcacg
     7261 tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg ttagcaccgc
     7321 aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat ggatttccgt
     7381 ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa aaaatggtgt
     7441 tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga tttttgaagc
     7501 aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc tggcctggtc
     7561 tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag tttcaatacc
     7621 ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact atatccgtaa
     7681 cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt agccatgttt
     7741 aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg
     7801 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     7861 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     7921 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     7981 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     8041 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     8101 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     8161 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     8221 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     8281 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     8341 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga
     8401 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
     8461 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
     8521 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     8581 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     8641 cacacacaag gctacttccc tgattggcag aac
//
