LOCUS       sequence-409107-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription plasmid for production of dSaCas9-KOX1 KRAB 
            mRNA for transcriptional activation (KRAB = Kruppel-associated box).
            Insert is derived from Addgene plasmid #106219..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6520)
  TITLE     In vitro transcription plasmid for production of dSaCas9-KOX1 KRAB 
            mRNA for transcriptional activation (KRAB = Kruppel-associated box).
            Insert is derived from Addgene plasmid #106219.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6520)
  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: 
            "Unpublished"
COMMENT     Sequence Label: dSaCas9-KRAB
FEATURES             Location/Qualifiers
     source          1..6520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             484..510
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             514..534
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3715..3762
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3796..3990
                     /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="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             4057..4077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4299..4315)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4299..4315)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4312..4334)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4323..4339
                     /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)."
     promoter        complement(join(4347..4353,4354..4371,4372..4377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4392..4413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4530..4547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4701..5289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4781..4800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5460..6251,6252..6320))
                     /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     6083..6102
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6321..6425)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6493..6511
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctaatacg
      421 actcactata aggaaataag agagaaaaga agagtaagaa gaaatatagc tagcgccacc
      481 atgtacccat acgatgttcc agattacgct gccccaaaga agaagcggaa ggtcggtatc
      541 cacggagtcc cagcagccaa gcggaactac atcctgggcc tggccatcgg catcaccagc
      601 gtgggctacg gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg
      661 ttcaaagagg ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg cgccagaagg
      721 ctgaagcggc ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac
      781 ctgctgaccg accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc
      841 ctgagccaga agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga
      901 agaggcgtgc acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa
      961 gagcagatca gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg
     1021 gaacggctga agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac
     1081 tacgtgaaag aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag
     1141 agcttcatcg acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct
     1201 ggcgagggca gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc
     1261 cactgcacct acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg
     1321 tacaacgccc tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg
     1381 gaatattacg agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc
     1441 ctgaagcaga tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg
     1501 accagcaccg gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt
     1561 accgcccgga aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg
     1621 accatctacc agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg
     1681 acccaggaag agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg
     1741 agcctgaagg ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc
     1801 gctatcttca accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag
     1861 atccccacca ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc
     1921 cagagcatca aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt
     1981 atcgagctgg cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag
     2041 aagcggaacc ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag
     2101 aacgccaagt acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg
     2161 tacagcctgg aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg
     2221 gaccacatca tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg
     2281 aagcaggaag aagccagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc
     2341 gacagcaaga tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag
     2401 ggcagaatca gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc
     2461 tccgtgcaga aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc
     2521 ctgatgaacc tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc
     2581 atcaatggcg gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac
     2641 aaggggtaca agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc
     2701 aaagagtgga agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa
     2761 aagcaggccg agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc
     2821 accccccacc agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg
     2881 gacaagaagc ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac
     2941 aagggcaaca ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag
     3001 ctgaaaaagc tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag
     3061 acctaccaga aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac
     3121 aagtactacg aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc
     3181 gtgatcaaga agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac
     3241 gactacccca acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac
     3301 gtgtacctgg acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa
     3361 aaagaaaact actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag
     3421 atcagcaacc aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac
     3481 ggcgagctgt atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac
     3541 atgatcgaca tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg
     3601 atcattaaga caatcgcctc caagacccag agcattaaga agtacagcac agacattctg
     3661 ggcaacctgt atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg
     3721 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccga tgctaagtca
     3781 ctgactgcct ggtcccggac actggtgacc ttcaaggatg tgtttgtgga cttcaccagg
     3841 gaggagtgga agctgctgga cactgctcag cagatcctgt acagaaatgt gatgctggag
     3901 aactataaga acctggtttc cttgggttat cagcttacta agccagatgt gatcctccgg
     3961 ttggagaagg gagaagagcc ctggctggtg gagagagaaa ttcaccaaga gacccatcct
     4021 gattcagaga ctgcatttga aatcaaatca tcagttccga aaaagaaacg caaagtttaa
     4081 ggcggccgct taattaagct gccttctgcg gggcttgcct tctggccatg cccttcttct
     4141 ctcccttgca cctgtacctc ttggtctttg aataaagcct gagtaggaag tctaraaaaa
     4201 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagggt cttcatcatc
     4261 gcctgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga
     4321 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4381 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4441 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4501 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4561 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4621 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4681 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4741 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4801 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4861 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4921 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4981 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5041 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5101 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5161 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5221 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5281 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5341 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5401 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5461 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5521 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5581 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5641 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5701 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5761 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5821 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5881 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5941 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6001 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6061 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6121 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6181 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6241 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6301 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6361 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6421 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6481 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
