LOCUS       sequence-351955-        8672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neomycin (bU6), Lkb1 (mU6) and non-targeting(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-sgLkb1_1st-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             124..304
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(366..954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(446..465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1125..1916,1917..1985))
                     /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     1748..1767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1986..2090)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2165..2184)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2356..2735
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2737..2935
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2890..2910
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2953..3133
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3180..3305
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3802..4035
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4220..4264
                     /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    4562..4679
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5416..5436
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5661..5681
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5832..5851
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6071..6570
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6106..6129)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6503..6522
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6574..6594
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6595..7623
                     /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    7679..8267
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7732..7752)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8150..8161)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8270..8286)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8271..8287)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag
       61 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt
      121 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      181 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      241 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      301 agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      361 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      421 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      481 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      541 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      601 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      661 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      721 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      781 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      841 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      901 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      961 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1021 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1081 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1141 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1201 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1261 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1321 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1381 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1441 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1501 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1561 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1621 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1681 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1741 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1801 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1861 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1921 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1981 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2041 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2101 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     2161 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     2221 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     2281 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     2341 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2401 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2461 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2521 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2581 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2641 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2701 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2761 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2821 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2881 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt
     2941 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     3001 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     3061 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     3121 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
     3181 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     3241 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     3301 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     3361 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     3421 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     3481 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     3541 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     3601 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccggc cgctgatctt
     3661 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     3721 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     3781 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     3841 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     3901 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     3961 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     4021 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     4081 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     4141 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     4201 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     4261 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     4321 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     4381 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     4441 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     4501 agtgaacgga tcggcactgc gtgcgccaat tctgcagaca aatggcagta ttcatccaca
     4561 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     4621 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     4681 gggtttatta cagggacagc agagatccag tttggttagt accgggcccg ctctagccct
     4741 gcaggaactg tttaaactgt cgtgaccgag cttgtctgcc cgcgcagtcc actagacaga
     4801 cgcgcagagg tcgggcgggc ctagggccaa tttcccatgg ttcccttcat ttgcatatat
     4861 gatgtaataa ggttatggag actattaaac ttagccctaa tcaaactata tgatgataat
     4921 gtgtgtggta caaaaggtca taacttatta tatactttga aacttaaaaa agggttacag
     4981 tttagtcacc ataactgtaa aattttttct attcttagct ttatatagtt cttgagaggc
     5041 catgtttatg gtcatggctg atgcaatgcg ggtttaagag ctaagctgga aacagcatag
     5101 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     5161 ttttccagtc tgactatcac gattgatccg acgcgccatc tctaggcccg cgccggcccc
     5221 ctcgcacgga cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa
     5281 tatttcctag taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt
     5341 aatactagct acattttaca tgataggctt ggatttctat aagagataca aatactaaat
     5401 tattatttta aaaaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt
     5461 ttgagactat aagtatccct tggagaacca ccttgttggt ggtgggccgc agtcacaagt
     5521 ttgagagcta agcagaaagc tgcatagcaa gttcaaataa ggctagtccg tacacaactt
     5581 gaaaaagtgg cagccgagtc ggctgctttt tttgctcgaa tctacactca gctatggcgc
     5641 gccccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
     5701 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     5761 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     5821 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     5881 tatatatctt gtggaaagcc agaaacatgg cgaggtattc ggctccgcgg tttcagagct
     5941 aagcacaaga gtgcatagca agttgaaata aggctagtcc gtttacaact tgaaaaagtg
     6001 gcacccgagt cgggtgcttt ttttgtttgg cgcgcctatg gcttaattaa catggcggcc
     6061 gcattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     6121 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     6181 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     6241 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     6301 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     6361 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     6421 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     6481 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     6541 ttctcctctt cctcatctcc gggacccggg atgcccaaga agaagaggaa ggtgtccaat
     6601 ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt
     6661 cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa
     6721 atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg
     6781 tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg
     6841 gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg
     6901 ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa
     6961 aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac
     7021 caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt
     7081 ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa
     7141 gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg
     7201 gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga
     7261 tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga
     7321 aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg
     7381 atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac
     7441 ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga
     7501 gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac
     7561 tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat
     7621 tagccatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa
     7681 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     7741 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     7801 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     7861 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     7921 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     7981 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     8041 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     8101 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     8161 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     8221 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     8281 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     8341 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     8401 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     8461 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
     8521 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     8581 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     8641 aggtagaaga agccaatgaa ggagagaaca cc
//
