LOCUS       sequence-352658-        8672 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 8672)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8672)
  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_1st
FEATURES             Location/Qualifiers
     source          1..8672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             53..233
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(295..883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(375..394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1054..1845,1846..1914))
                     /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     1677..1696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1915..2019)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2094..2113)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2285..2664
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2666..2864
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2819..2839
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2882..3062
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3109..3234
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3731..3964
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4149..4193
                     /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    4491..4608
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5345..5365
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5590..5610
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5761..5780
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6000..6499
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6035..6058)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6432..6451
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6503..6523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6524..7552
                     /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    7608..8196
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7661..7681)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8079..8090)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8199..8215)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8200..8216)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
       61 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      121 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      181 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg
      241 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      301 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      361 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      421 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      481 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      541 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      601 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      661 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      721 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      781 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      841 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      901 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
      961 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1021 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1081 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1141 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1201 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1261 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1321 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1381 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1441 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1501 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1561 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1621 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1681 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1741 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1801 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     1861 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     1921 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1981 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2041 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     2101 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     2161 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     2221 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     2281 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2341 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2401 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2461 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2521 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     2581 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     2641 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     2701 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     2761 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     2821 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac
     2881 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     2941 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     3001 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     3061 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     3121 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     3181 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     3241 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     3301 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     3361 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     3421 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     3481 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     3541 caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc agacctggag
     3601 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     3661 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     3721 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     3781 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     3841 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     3901 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     3961 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     4021 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     4081 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     4141 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     4201 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     4261 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     4321 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     4381 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     4441 cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga
     4501 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     4561 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     4621 agggacagca gagatccagt ttggttagta ccgggcccgc tctagccctg caggaactgt
     4681 ttaaactgtc gtgaccgagc ttgtctgccc gcgcagtcca ctagacagac gcgcagaggt
     4741 cgggcgggcc tagggccaat ttcccatggt tcccttcatt tgcatatatg atgtaataag
     4801 gttatggaga ctattaaact tagccctaat caaactatat gatgataatg tgtgtggtac
     4861 aaaaggtcat aacttattat atactttgaa acttaaaaaa gggttacagt ttagtcacca
     4921 taactgtaaa attttttcta ttcttagctt tatatagttc ttgagaggcc atgtttatgg
     4981 tcatggctga tgcaatgcgg gtttaagagc taagctggaa acagcatagc aagtttaaat
     5041 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttccagtct
     5101 gactatcacg attgatccga cgcgccatct ctaggcccgc gccggccccc tcgcacggac
     5161 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
     5221 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
     5281 cattttacat gataggcttg gatttctata agagatacaa atactaaatt attattttaa
     5341 aaaacagcac aaaaggaaac tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata
     5401 agtatccctt ggagaaccac cttgttggcg aggtattcgg ctccgcggtt tgagagctaa
     5461 gcagaaagct gcatagcaag ttcaaataag gctagtccgt acacaacttg aaaaagtggc
     5521 agccgagtcg gctgcttttt ttgctcgaat ctacactcag ctatggcgcg ccccaaggtc
     5581 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     5641 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     5701 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     5761 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     5821 tggaaagcca gaaacatggt ggtgggccgc agtcacaagt ttcagagcta agcacaagag
     5881 tgcatagcaa gttgaaataa ggctagtccg tttacaactt gaaaaagtgg cacccgagtc
     5941 gggtgctttt tttgtttggc gcgcctatgg cttaattaac atggcggccg cattctaccg
     6001 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     6061 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     6121 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     6181 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     6241 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     6301 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     6361 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     6421 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     6481 ctcatctccg ggacccggga tgcccaagaa gaagaggaag gtgtccaatt tactgaccgt
     6541 acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc gcaagaacct
     6601 gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc
     6661 cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt ttcccgcaga
     6721 acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg cagtaaaaac
     6781 tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc tgccacgacc
     6841 aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa acgttgatgc
     6901 cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc aggttcgttc
     6961 actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc tggggattgc
     7021 ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag atatctcacg
     7081 tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg ttagcaccgc
     7141 aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat ggatttccgt
     7201 ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa aaaatggtgt
     7261 tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga tttttgaagc
     7321 aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc tggcctggtc
     7381 tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag tttcaatacc
     7441 ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact atatccgtaa
     7501 cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt agccatgttt
     7561 aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg
     7621 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     7681 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     7741 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     7801 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     7861 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     7921 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     7981 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     8041 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     8101 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     8161 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga
     8221 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
     8281 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
     8341 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     8401 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     8461 cacacacaag gctacttccc tgattggcag aactacacac cagggccagg gatcagatat
     8521 ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa
     8581 gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac
     8641 ccggagagag aagtattaga gtggaggttt ga
//
