LOCUS       sequence-350026-        8248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Sik3-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-sgSik3_2nd-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             210..390
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(452..1040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(532..551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1211..2002,2003..2071))
                     /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     1834..1853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2072..2176)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2251..2270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2442..2821
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2823..3021
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2976..2996
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3039..3219
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3266..3391
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3888..4121
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4306..4350
                     /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    4648..4765
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5078..5098
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5323..5343
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5494..5513
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5733..6232
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5768..5791)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6165..6184
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6236..6256
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6257..7285
                     /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    7341..7929
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7394..7414)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7812..7823)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7932..7948)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7933..7949)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
       61 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
      121 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
      181 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
      241 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      301 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      361 ccttttagtc agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca
      421 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      481 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      541 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      601 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      661 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
      721 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      781 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
      841 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
      901 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
      961 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1021 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1081 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1141 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1201 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     1261 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     1321 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     1381 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     1441 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     1501 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     1561 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     1621 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     1681 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     1741 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     1801 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     1861 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     1921 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     1981 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     2041 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     2101 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2161 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     2221 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2281 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     2341 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     2401 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     2461 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     2521 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     2581 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     2641 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     2701 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     2761 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     2821 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     2881 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     2941 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     3001 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
     3061 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     3121 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     3181 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     3241 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     3301 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     3361 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     3421 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     3481 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     3541 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     3601 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     3661 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     3721 acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     3781 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     3841 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     3901 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     3961 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     4021 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     4081 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     4141 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     4201 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     4261 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     4321 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     4381 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     4441 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     4501 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     4561 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     4621 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     4681 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     4741 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     4801 gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgatc cgacgcgcca
     4861 tctctaggcc cgcgccggcc ccctcgcacg gacttgtggg agaagctcgg ctactcccct
     4921 gccccggtta atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa
     4981 agacagataa tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct
     5041 ataagagata caaatactaa attattattt taaaaaacag cacaaaagga aactcaccct
     5101 aactgtaaag taattgtgtg ttttgagact ataagtatcc cttggagaac caccttgttg
     5161 gttcaaacag atcgtcacag gtttgagagc taagcagaaa gctgcatagc aagttcaaat
     5221 aaggctagtc cgtacacaac ttgaaaaagt ggcagccgag tcggctgctt tttttgctcg
     5281 aatctacact cagctatggc gcgccccaag gtcgggcagg aagagggcct atttcccatg
     5341 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     5401 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     5461 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     5521 aagtatttcg atttcttggc tttatatatc ttgtggaaag ccagaaacat ggcgaggtat
     5581 tcggctccgc ggtttcagag ctaagcacaa gagtgcatag caagttgaaa taaggctagt
     5641 ccgtttacaa cttgaaaaag tggcacccga gtcgggtgct ttttttgttt ggcgcgccta
     5701 tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt tcccaaggca
     5761 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     5821 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     5881 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     5941 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     6001 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     6061 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     6121 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     6181 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg ggatgcccaa
     6241 gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg cattaccggt
     6301 cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc
     6361 gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg
     6421 caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct
     6481 atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa
     6541 catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt
     6601 tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc
     6661 gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca
     6721 ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac gtatagccga
     6781 aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca
     6841 tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg
     6901 ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa
     6961 ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct
     7021 atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa
     7081 ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc
     7141 gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac
     7201 caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt
     7261 gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac taggtacaat
     7321 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     7381 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     7441 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     7501 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     7561 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     7621 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     7681 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     7741 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     7801 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     7861 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     7921 cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca
     7981 agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag
     8041 gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct
     8101 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     8161 cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg
     8221 cagaactaca caccagggcc agggatca
//
