LOCUS       sequence-363978-        8673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sik1(bU6), Sik2 (mU6) and Sik3 (hU6) gRNAs and 
            Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8673)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8673)
  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-sgSik1_1st-mU6-sgSik2-hU6-sgSik3_1st
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             179..359
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(421..1009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(501..520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1180..1971,1972..2040))
                     /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     1803..1822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2041..2145)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2220..2239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2411..2790
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2792..2990
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2945..2965
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3008..3188
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3235..3360
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3857..4090
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4275..4319
                     /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    4617..4734
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5470..5490
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5716..5736
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5887..5906
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6127..6626
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6162..6185)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6559..6578
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6630..6650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6651..7679
                     /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    7735..8323
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7788..7808)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8206..8217)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8326..8342)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8327..8343)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
       61 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
      121 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
      181 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      241 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      301 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2161 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     2221 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     2281 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     2341 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     2401 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2461 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2521 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2581 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2641 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2701 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2761 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2821 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2881 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2941 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc
     3001 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     3061 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     3121 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     3181 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     3241 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     3301 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     3361 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     3421 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     3481 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     3541 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     3601 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     3661 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac
     3721 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     3781 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     3841 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     3901 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     3961 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     4021 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     4081 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     4141 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     4201 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     4261 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     4321 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     4381 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     4441 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     4501 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     4561 acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt
     4621 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     4681 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     4741 tattacaggg acagcagaga tccagtttgg ttagtaccgg gcccgctcta gccctgcagg
     4801 aactgtttaa actgtcgtga ccgagcttgt ctgcccgcgc agtccactag acagacgcgc
     4861 agaggtcggg cgggcctagg gccaatttcc catggttccc ttcatttgca tatatgatgt
     4921 aataaggtta tggagactat taaacttagc cctaatcaaa ctatatgatg ataatgtgtg
     4981 tggtacaaaa ggtcataact tattatatac tttgaaactt aaaaaagggt tacagtttag
     5041 tcaccataac tgtaaaattt tttctattct tagctttata tagttcttga gaggccatgt
     5101 ttatggatca tgtcggagtt cagtggttta agagctaagc tggaaacagc atagcaagtt
     5161 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc
     5221 agtctgacta tcacgattga tccgacgcgc catctctagg cccgcgccgg ccccctcgca
     5281 cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat ataatatttc
     5341 ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct ttttaatact
     5401 agctacattt tacatgatag gcttggattt ctataagaga tacaaatact aaattattat
     5461 tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg tgttttgaga
     5521 ctataagtat cccttggaga accaccttgt tggtgataca gtttaatgat gtggtttgag
     5581 agctaagcag aaagctgcat agcaagttca aataaggcta gtccgtacac aacttgaaaa
     5641 agtggcagcc gagtcggctg ctttttttgc tcgaatctac actcagctat ggcgcgcccc
     5701 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
     5761 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     5821 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     5881 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     5941 atcttgtgga aagccagaaa catggtggca gagaaggaag ctcgagtttc agagctaagc
     6001 acaagagtgc atagcaagtt gaaataaggc tagtccgttt acaacttgaa aaagtggcac
     6061 ccgagtcggg tgcttttttt gtttggcgcg cctatggctt aattaacatg gcggccgcat
     6121 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     6181 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     6241 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     6301 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     6361 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     6421 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     6481 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     6541 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     6601 cctcttcctc atctccggga cccgggatgc ccaagaagaa gaggaaggtg tccaatttac
     6661 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
     6721 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
     6781 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
     6841 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
     6901 taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
     6961 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
     7021 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
     7081 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
     7141 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
     7201 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
     7261 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
     7321 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     7381 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     7441 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     7501 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     7561 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata
     7621 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagc
     7681 catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa
     7741 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7801 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7861 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     7921 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     7981 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     8041 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     8101 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     8161 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     8221 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     8281 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
     8341 cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     8401 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     8461 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     8521 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     8581 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat
     8641 cagatatcca ctgacctttg gatggtgcta caa
//
