LOCUS       sequence-350031-        8250 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 8250)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8250)
  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-sgSik3_1st
FEATURES             Location/Qualifiers
     source          1..8250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..78
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             130..150
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             151..1179
                     /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    1235..1823
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1288..1308)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1706..1717)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1826..1842)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1827..1843)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2352..2532
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2594..3182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2674..2693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3353..4144,4145..4213))
                     /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     3976..3995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4214..4318)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4393..4412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4584..4963
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4965..5163
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5118..5138
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5181..5361
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5408..5533
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6030..6263
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6448..6492
                     /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    6790..6907
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     7220..7240
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     7466..7486
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7637..7656
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        join(7877..8250,1..126)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7912..7935)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
       61 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccggga
      121 cccgggatgc ccaagaagaa gaggaaggtg tccaatttac tgaccgtaca ccaaaatttg
      181 cctgcattac cggtcgatgc aacgagtgat gaggttcgca agaacctgat ggacatgttc
      241 agggatcgcc aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg
      301 tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt
      361 cgcgattatc ttctatatct tcaggcgcgc ggtctggcag taaaaactat ccagcaacat
      421 ttgggccagc taaacatgct tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat
      481 gctgtttcac tggttatgcg gcggatccga aaagaaaacg ttgatgccgg tgaacgtgca
      541 aaacaggctc tagcgttcga acgcactgat ttcgaccagg ttcgttcact catggaaaat
      601 agcgatcgct gccaggatat acgtaatctg gcatttctgg ggattgctta taacaccctg
      661 ttacgtatag ccgaaattgc caggatcagg gttaaagata tctcacgtac tgacggtggg
      721 agaatgttaa tccatattgg cagaacgaaa acgctggtta gcaccgcagg tgtagagaag
      781 gcacttagcc tgggggtaac taaactggtc gagcgatgga tttccgtctc tggtgtagct
      841 gatgatccga ataactacct gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct
      901 gccaccagcc agctatcaac tcgcgccctg gaagggattt ttgaagcaac tcatcgattg
      961 atttacggcg ctaaggatga ctctggtcag agatacctgg cctggtctgg acacagtgcc
     1021 cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt caataccgga gatcatgcaa
     1081 gctggtggct ggaccaatgt aaatattgtc atgaactata tccgtaacct ggatagtgaa
     1141 acaggggcaa tggtgcgcct gctggaagat ggcgattagc catgtttaag ggttccggtt
     1201 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     1261 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     1321 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     1381 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     1441 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     1501 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     1561 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     1621 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     1681 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     1741 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     1801 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     1861 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     1921 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     1981 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     2041 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     2101 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     2161 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     2221 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     2281 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     2341 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     2401 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     2461 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     2521 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2581 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2641 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2701 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2761 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2821 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2881 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2941 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3001 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3061 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3121 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3181 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3241 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3301 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3361 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3421 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3481 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3541 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3601 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3661 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3721 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3781 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3841 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3901 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3961 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4021 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4081 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4141 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4201 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4261 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4321 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     4381 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     4441 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     4501 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     4561 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     4621 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     4681 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     4741 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     4801 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     4861 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     4921 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     4981 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     5041 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     5101 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     5161 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     5221 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5281 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     5341 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     5401 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     5461 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     5521 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     5581 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     5641 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     5701 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     5761 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     5821 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     5881 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     5941 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     6001 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     6061 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     6121 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     6181 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     6241 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     6301 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     6361 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     6421 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     6481 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     6541 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     6601 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     6661 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     6721 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     6781 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     6841 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     6901 aaattttcgg gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct
     6961 ctagccctgc aggaactgtt taaactgtga tccgacgcgc catctctagg cccgcgccgg
     7021 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
     7081 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
     7141 ttttaatact agctacattt tacatgatag gcttggattt ctataagaga tacaaatact
     7201 aaattattat tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
     7261 tgttttgaga ctataagtat cccttggaga accaccttgt tggtctggag aagatctacc
     7321 ggggtttgag agctaagcag aaagctgcat agcaagttca aataaggcta gtccgtacac
     7381 aacttgaaaa agtggcagcc gagtcggctg ctttttttgc tcgaatctac actcagctat
     7441 ggcgcgcccc aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     7501 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     7561 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     7621 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     7681 ggctttatat atcttgtgga aagccagaaa catggtggca gagaaggaag ctcgagtttc
     7741 agagctaagc acaagagtgc atagcaagtt gaaataaggc tagtccgttt acaacttgaa
     7801 aaagtggcac ccgagtcggg tgcttttttt gtttggcgcg cctatggctt aattaacatg
     7861 gcggccgcat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     7921 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     7981 acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct
     8041 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     8101 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     8161 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     8221 cagaggctgg gaaggggtgg gtccgggggc
//
