LOCUS       sequence-349895-        8249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Sik1-targeting (mU6) and a non-targeting (hU6) gRNAs and
            Cre recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8249)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8249)
  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-sgSik1_2nd-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             214..394
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(456..1044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(536..555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1215..2006,2007..2075))
                     /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     1838..1857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2076..2180)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2255..2274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2446..2825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2827..3025
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2980..3000
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3043..3223
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3270..3395
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3892..4125
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4310..4354
                     /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    4652..4769
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5082..5102
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5328..5348
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5499..5518
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5738..6237
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5773..5796)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6170..6189
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6241..6261
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6262..7290
                     /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    7346..7934
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7399..7419)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7817..7828)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7937..7953)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7938..7954)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag
       61 gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg
      121 atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat
      181 cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga
      241 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      301 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      361 agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag
      421 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      481 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      541 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      601 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      661 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      721 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      781 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      841 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
      901 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      961 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     1021 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     1081 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     1141 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     1201 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     1261 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     1321 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     1381 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1441 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1501 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     1561 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1621 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1681 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1741 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1801 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     1861 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1921 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1981 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     2041 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     2101 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     2161 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     2221 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2281 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     2341 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     2401 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     2461 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2521 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2581 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2641 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2701 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2761 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2821 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     2881 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     2941 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     3001 tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac
     3061 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     3121 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     3181 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
     3241 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
     3301 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
     3361 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
     3421 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
     3481 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     3541 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     3601 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     3661 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     3721 ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     3781 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     3841 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     3901 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     3961 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     4021 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     4081 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     4141 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     4201 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     4261 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     4321 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     4381 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     4441 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     4501 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     4561 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
     4621 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     4681 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     4741 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     4801 tttggttagt accgggcccg ctctagccct gcaggaactg tttaaactgt gatccgacgc
     4861 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc
     4921 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     4981 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     5041 ttctataaga gatacaaata ctaaattatt attttaaaaa acagcacaaa aggaaactca
     5101 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     5161 gttggtctgg agaagatcta ccggggtttg agagctaagc agaaagctgc atagcaagtt
     5221 caaataaggc tagtccgtac acaacttgaa aaagtggcag ccgagtcggc tgcttttttt
     5281 gctcgaatct acactcagct atggcgcgcc ccaaggtcgg gcaggaagag ggcctatttc
     5341 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     5401 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     5461 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     5521 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaagccaga aacatggcga
     5581 ggtattcggc tccgcggttt cagagctaag cacaagagtg catagcaagt tgaaataagg
     5641 ctagtccgtt tacaacttga aaaagtggca cccgagtcgg gtgctttttt tgtttggcgc
     5701 gcctatggct taattaacat ggcggccgca ttctaccggg taggggaggc gcttttccca
     5761 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     5821 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     5881 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     5941 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     6001 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     6061 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     6121 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     6181 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg acccgggatg
     6241 cccaagaaga agaggaaggt gtccaattta ctgaccgtac accaaaattt gcctgcatta
     6301 ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc
     6361 caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca
     6421 tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat
     6481 cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag
     6541 ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca
     6601 ctggttatgc ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct
     6661 ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc
     6721 tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata
     6781 gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta
     6841 atccatattg gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa ggcacttagc
     6901 ctgggggtaa ctaaactggt cgagcgatgg atttccgtct ctggtgtagc tgatgatccg
     6961 aataactacc tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc
     7021 cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt gatttacggc
     7081 gctaaggatg actctggtca gagatacctg gcctggtctg gacacagtgc ccgtgtcgga
     7141 gccgcgcgag atatggcccg cgctggagtt tcaataccgg agatcatgca agctggtggc
     7201 tggaccaatg taaatattgt catgaactat atccgtaacc tggatagtga aacaggggca
     7261 atggtgcgcc tgctggaaga tggcgattag ccatgtttaa gggttccggt tccactaggt
     7321 acaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     7381 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     7441 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     7501 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     7561 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     7621 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     7681 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     7741 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     7801 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     7861 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     7921 ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa
     7981 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg
     8041 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg
     8101 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     8161 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     8221 attggcagaa ctacacacca gggccaggg
//
