LOCUS       sequence-449902-        8816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of rTetR(SE) fused to ZNF10 KRAB and 
            mTurquoise-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8816)
  AUTHORS   Sinha J, Nickels JF, Thurm AR, Ludwig CH, Archibald BN, Hinks MM, 
            Wan J, Fang D, Bintu L
  TITLE     The H3.3 K36M oncohistone disrupts the establishment of epigenetic 
            memory through loss of DNA methylation.
  JOURNAL   bioRxiv [Preprint]. 2023 Oct 14:2023.10.13.562147. doi: 
            10.1101/2023.10.13.562147.
  PUBMED    37873347
REFERENCE   2  (bases 1 to 8816)
  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"; journalName: "bioRxiv 
            [Preprint]. 2023 Oct 14:2023.10.13.562147. doi: 
            10.1101/2023.10.13.562147."
COMMENT     Sequence Label: pLenti-rTetR(SE)-KRAB-2A-mTurquoise
FEATURES             Location/Qualifiers
     source          1..8816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             651..831
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     1340..1356
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1341..1357
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1360..1948
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1466..1477
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1875..1895
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    2004..2037
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2060..2080)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(2099..2809,2810..2812,2813..2815))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2010)"
                     /label=mTurquoise
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2141..2162)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2490..2509
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2749..2770
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2816..2869)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2879..3064)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             complement(3113..3178)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     intron          3819..4757
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3842..3862)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(4758..4969)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    5092..5209
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5507..5551)
                     /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    5736..5969
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6466..6591
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6638..6818
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6836..7034)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6861..6881)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7036..7415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     7587..7606
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7681..7785
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7786..7854,7855..8646)
                     /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(8004..8023)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgc tgctagagat
      661 tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac acaacagacg
      721 ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca gtgggttccc
      781 tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc cagtacagtc
      841 cggatgcagc tctcgggcca tgtgatgaaa tgctaggcgg ctgtcaaacc tccactctaa
      901 tacttctctc tccgggtcat ccatcccatg caggctcaca gggtgtaaca agcgggtgtt
      961 ctctccttca ttggcttctt ctaccttctc ttgctcaact ggtactagct tgtagcacca
     1021 tccaaaggtc agtggatatc tgatccctgg ccctggtgtg tagttctgcc aatcagggaa
     1081 gtagccttgt gtgtggtaga tccacagatc aaggatatct tgtcttcgtt gggagtgaat
     1141 tagcccttcc agtcccccct tttcttttaa aaagtggcta agatctacag ctgccttgta
     1201 agtcattggt cttaaaggta cctgaggtgt gactggaaaa cccacctcct cctcctcttg
     1261 tgcttctagc caggcacaat cagcattggt agctgctgta ttgctacttg tgattgctcc
     1321 atgtttttct aggtctcgat cgaggtcgac ggtatcgatg cggggaggcg gcccaaaggg
     1381 agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca
     1441 ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag aaggacgtcc
     1501 cgcgcagaat ccaggtggca acacaggcga gcagccaagg aaaggacgat gatttccccg
     1561 acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc
     1621 aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc ccggaaagga
     1681 gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac acagtgcaca
     1741 ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc aaccaggatt
     1801 tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta
     1861 aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt
     1921 cacaaatttt gtaatccaga ggttgattat cgataagctt gatatcgaat tgtacctagt
     1981 ggaaccggaa cccttaaaca tgtataactt cgtataatgt atgctatacg aagttattag
     2041 gtccctcgac gaattcttat acctttctct tcttttttgg gctaccgctg ccgctaccct
     2101 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     2161 tgtgatcgcg cttctcgttg gggtctttgc tcagcttgga ctgggtgctc aggtagtggt
     2221 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     2281 gctgcacgcc gccgtcctcg atgttgtggc ggatcttgaa gttggccttg atgccgttct
     2341 tctgcttgtc ggcggtgata tagacgttgt cgctgatgta gttgtactcc agcttgtgcc
     2401 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     2461 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     2521 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     2581 tgtggtcggg gtagcgggcg aagcactgca cgccccagga cagggtggtc acgagggtgg
     2641 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     2701 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     2761 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accattgggc
     2821 caggattctc ctcgacatct ccgcaagtta ggaggcttcc ccttccctct ccagatccgg
     2881 gctcttctcc cttctccaac cggaggatca catctggctt agtaagctga taacccaagg
     2941 aaaccaggtt cttatagttc tccagcatca catttctgta cacgatctgc tgagcagtgt
     3001 ccagcagctt ccactcctcc ctggtgaagt ccacaaatac atccttgaag gtcaccagtg
     3061 tccgggacca ggcagttagt gacttagcat cggagcctga tccggatcca cccttgtcat
     3121 cgtcatcctt gtagtcgatg tcatgatctt tataatcacc gtcatggtct ttgtagtcac
     3181 cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa
     3241 ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtttc agaagtgggg
     3301 gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgttcct
     3361 ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca
     3421 gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct
     3481 ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg agagcacagc
     3541 ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgggttcc agggggcagg
     3601 agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg gcccgcttgt
     3661 tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg
     3721 tcgtcaggcc ttcgataccg acaccattga gtaattccag agctccgttt attattttgc
     3781 tcttgtccag tctagacatg gtggctagct cacgacacct gaaatggaag aaaaaaactt
     3841 tgaaccactg tctgaggctt gagaatgaac caagatccaa actcaaaaag ggcaaattcc
     3901 aaggagaatt acatcaagtg ccaagctggc ctaacttcag tctccaccca ctcagtgtgg
     3961 ggaaactcca tcgcataaaa cccctccccc caacctaaag acgacgtact ccaaaagcgc
     4021 gagaactaat cgaggtgcct ggacggcgcc cggtactccg tggagtcaca tgaagcgacg
     4081 gctgaggacg gaaaggccct tttcctttgt gtgggtgact cacccgcccg ctctcccgag
     4141 cgccgcgtcc tccattttga gctccctgca gcagggccgg gaagcggcca tctttccgct
     4201 cacgcaactg gtgccgaccg ggccagcctt gccgcccagg gcggggcgat acacggcggc
     4261 gcgaggccag gcaccagagc aggccggcca gcttgagact acccccgtcc gattctcggt
     4321 ggccgcgctc gcaggccccg cctcgccgaa catgtgcgct gggacgcacg ggccccgtcg
     4381 ccgcccgcgg ccccaaaaac cgaaatacca gtgtgcagat cttggcccgc atttacaaga
     4441 ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa aattttaaat ggctagagac
     4501 ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga ttcgcacgcg gcggccccag
     4561 cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg ctccttaagc gcaaggcctc
     4621 gaactctccc acccacttcc aacccgaagc tcgggatcaa gaatcacgta ctgcagccag
     4681 gtggaagtaa ttcaaggcac gcaagggcca taacccgtaa agaggccagg cccgcgggaa
     4741 ccacacacgg cacttacctg tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg
     4801 gcgactactg cacttatata cggttctccc ccaccctcgg gaaaaaggcg gagccagtac
     4861 acgacatcac tttcccagtt taccccgcgc caccttctct aggcaccggt tcaattgccg
     4921 acccctcccc ccaacttctc ggggactgtg ggcgatgtgc gctctgccca ctgacgggca
     4981 catgcatggc ggtaatacgg ttatccacgc ggccgcctga gtcaaatggg atctctagag
     5041 cgggcccggt actaaccaaa ctggatctct gctgtccctg taataaaccc gaaaattttg
     5101 aatttttgta atttgttttt gtaattcttt agtttgtatg tctgttgcta ttatgtctac
     5161 tattctttcc cctgcactgt accccccaat cccccctttt cttttaaaat tgtggatgaa
     5221 tactgccatt tgtctgcaga attggcgcac gcagtgccga tccgttcact aatcgaatgg
     5281 atctgtctct gtctctctct ccaccttctt cttctattcc ttcgggcctg tcgggtcccc
     5341 tcggggttgg gaggtgggtc tgaaacgata atggtgaata tccctgccta actctattca
     5401 ctatagaaag tacagcaaaa actattctta aacctaccaa gcctcctact atcattatga
     5461 ataattttat ataccacagc caatttgtta tgttaaacca attccacaaa cttgcccatt
     5521 tatctaattc caataattct tgttcattct tttcttgctg gttttgcgat tcttcaatta
     5581 aggagtgtat taagcttgtg taattgttaa tttctctgtc ccactccatc caggtcgtgt
     5641 gattccaaat ctgttccaga gatttattac tccaactagc attccaaggc acagcagtgg
     5701 tgcaaatgag ttttccagag caaccccaaa tccccaggag ctgttgatcc tttaggtatc
     5761 tttccacagc caggattctt gcctggagct gcttgatgcc ccagactgtg agttgcaaca
     5821 gatgctgttg cgcctcaata gccctcagca aattgttctg ctgctgcact ataccagaca
     5881 ataattgtct ggcctgtacc gtcagcgtca ttgacgctgc gcccatagtg cttcctgctg
     5941 ctcccaagaa cccaaggaac aaagctccta ttcccactgc tcttttttct ctctgcacca
     6001 ctcttctctt tgccttggtg ggtgctactc ctaatggttc aatttttact actttatatt
     6061 tatataattc acttctccaa ttgtccctca tatctcctcc tccaggtctg aagatcagcg
     6121 gccggccgct tgctgtgcgg tggtcttact tttgttttgc tcttcctcta tcttgtctaa
     6181 agcttccttg gtgtctttta tctctatcct ttgatgcaca caatagaggg ttgctactgt
     6241 attatataat gatctaagtt cttctgatcc tgtctgaagg gatggttgta gctgtcccag
     6301 tatttgtcta cagccttctg atgtttctaa caggccagga ttaactgcga atcgttctag
     6361 ctccctgctt gcccatacta tatgttttaa tttatatttt ttctttcccc ctggccttaa
     6421 ccgaattttt tcccatcgcg atctaattct cccccgctta atactgacgc tctcgcaccc
     6481 atctctctcc ttctagcctc cgctagtcaa aatttttggc gtactcacca gtcgccgccc
     6541 ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg
     6601 tttccctttc gctttcaagt ccctgttcgg gcgccactgc tagagatttt ccacactgac
     6661 taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc acacactact
     6721 tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag ttagccagag
     6781 agctcccagg ctcagatctg gtctaaccag agagacccag tacaggcaaa acgcgctgct
     6841 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
     6901 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
     6961 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
     7021 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
     7081 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     7141 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     7201 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     7261 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     7321 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     7381 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     7441 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt cggtcaagcc
     7501 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     7561 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     7621 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     7681 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     7741 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     7801 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     7861 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     7921 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     7981 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     8041 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     8101 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     8161 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     8221 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     8281 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     8341 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     8401 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     8461 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     8521 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     8581 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     8641 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     8701 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     8761 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
