LOCUS       sequence-349897-        8249 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 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-sgSik3_1st-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             192..372
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(434..1022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(514..533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1193..1984,1985..2053))
                     /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     1816..1835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2054..2158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2233..2252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2424..2803
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2805..3003
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2958..2978
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3021..3201
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3248..3373
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3870..4103
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4288..4332
                     /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    4630..4747
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5060..5080
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5306..5326
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5477..5496
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5716..6215
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5751..5774)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6148..6167
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6219..6239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6240..7268
                     /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    7324..7912
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7377..7397)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7795..7806)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7915..7931)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7916..7932)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
       61 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
      121 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
      181 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
      241 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
      301 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
      361 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      841 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1081 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1141 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1201 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1261 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1321 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1381 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1441 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1501 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1561 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1621 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1681 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1741 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1801 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1861 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1921 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1981 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     2041 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2101 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     2161 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     2221 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     2281 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     2341 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     2401 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     2461 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     2521 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     2581 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2641 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2701 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2761 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2821 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2881 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2941 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     3001 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     3061 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     3121 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga ctcttttagt
     3181 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     3241 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     3301 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     3361 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     3421 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     3481 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     3541 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     3601 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     3661 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     3721 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     3781 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     3841 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     3901 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     3961 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     4021 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     4081 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     4141 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     4201 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     4261 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     4321 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     4381 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     4441 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     4501 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     4561 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     4621 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     4681 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     4741 aaattttcgg gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct
     4801 ctagccctgc aggaactgtt taaactgtga tccgacgcgc catctctagg cccgcgccgg
     4861 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
     4921 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
     4981 ttttaatact agctacattt tacatgatag gcttggattt ctataagaga tacaaatact
     5041 aaattattat tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
     5101 tgttttgaga ctataagtat cccttggaga accaccttgt tggtggcaga gaaggaagct
     5161 cgagtttgag agctaagcag aaagctgcat agcaagttca aataaggcta gtccgtacac
     5221 aacttgaaaa agtggcagcc gagtcggctg ctttttttgc tcgaatctac actcagctat
     5281 ggcgcgcccc aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     5341 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     5401 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     5461 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     5521 ggctttatat atcttgtgga aagccagaaa catggcgagg tattcggctc cgcggtttca
     5581 gagctaagca caagagtgca tagcaagttg aaataaggct agtccgttta caacttgaaa
     5641 aagtggcacc cgagtcgggt gctttttttg tttggcgcgc ctatggctta attaacatgg
     5701 cggccgcatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     5761 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     5821 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     5881 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     5941 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     6001 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     6061 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     6121 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     6181 cgctgttctc ctcttcctca tctccgggac ccgggatgcc caagaagaag aggaaggtgt
     6241 ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg
     6301 aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct
     6361 ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga
     6421 aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg
     6481 gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt catcgtcggt
     6541 ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa
     6601 aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt
     6661 tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg
     6721 catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg
     6781 ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa
     6841 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg
     6901 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg
     6961 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg
     7021 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga
     7081 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg
     7141 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca
     7201 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg
     7261 gcgattagcc atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc
     7321 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7381 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7441 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7501 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     7561 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     7621 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     7681 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     7741 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     7801 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     7861 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     7921 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     7981 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     8041 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     8101 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     8161 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     8221 gccagggatc agatatccac tgacctttg
//
