LOCUS       sequence-267202-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9-mediated inactivation of HOTTIP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  AUTHORS   Luo H, Zhu G, Xu J, Lai Q, Yan B, Guo Y, Fung TK, Zeisig BB, Cui Y, 
            Zha J, Cogle C, Wang F, Xu B, Yang FC, Li W, So CWE, Qiu Y, Xu M, 
            Huang S
  TITLE     HOTTIP lncRNA Promotes Hematopoietic Stem Cell Self-Renewal Leading 
            to AML-like Disease in Mice.
  JOURNAL   Cancer Cell. 2019 Dec 9;36(6):645-659.e8. doi: 
            10.1016/j.ccell.2019.10.011. Epub 2019 Nov 27.
  PUBMED    31786140
REFERENCE   2  (bases 1 to 7410)
  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"; doi: 
            "10.1016/j.ccell.2019.10.011"; journalName: "Cancer Cell"; date: 
            "2019-12-9- 9"; volume: "36"; issue: "6"; pages: "645-659.e8"
COMMENT     Sequence Label: pLKO5-SgHottip-GFP-CRISPRi
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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"
     promoter        1718..1966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1718..1738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1889..1908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1987..2062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2110..2227
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2296..2507
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2527..2536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2533..2535,2536..2538,2539..3252)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2533..2535,2536..2538,2539..3249)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2578..2599)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2839..2858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3186..3207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3268..3856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3321..3341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3739..3750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3928..4161
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4239..4360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4276..4295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4330..4349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4400..4535
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4555..4574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4556..4574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4584..4601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4584..4600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4593..4615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4742..5197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4829..4848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5039..5060
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5223..5327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5328..5396,5397..6188)
                     /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     complement(5546..5565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6359..6947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6848..6867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7101..7118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7235..7256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7271..7276,7277..7294,7295..7301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7309..7325
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7314..7336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7333..7349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7333..7349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7368..7388
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7370..7388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     1741 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     1801 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     1861 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     1921 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcgc gggtctgggc
     1981 cccactgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2041 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     2101 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2161 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2221 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     2281 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2341 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2401 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2461 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     2521 ggatccgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2581 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2641 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     2701 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     2761 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     2821 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2881 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2941 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3001 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3061 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3121 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3181 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3241 ctgtacaagt aaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga
     3301 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3361 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3421 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3481 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3541 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3601 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3661 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3721 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3781 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3841 tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta
     3901 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     3961 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     4021 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     4081 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     4141 cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt
     4201 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
     4261 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4321 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat
     4381 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4441 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4501 attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat
     4561 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4621 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     4681 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4741 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4801 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4861 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4921 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4981 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5041 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5101 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5161 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     5221 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5281 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5341 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5401 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5461 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5521 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5581 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5641 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5701 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5761 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5821 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5881 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5941 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6001 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6061 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6121 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6181 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6241 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6301 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6361 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6421 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6481 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6541 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6601 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6661 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6721 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6781 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6841 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6901 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6961 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7021 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7081 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     7141 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     7201 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     7261 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     7321 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
     7381 actaaaggga acaaaagctg gagctgcaag
//
