LOCUS       sequence-183593-        7503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription template (T7 promoter) for Kdm6b catalytic 
            domain mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7503)
  AUTHORS   Inoue A, Jiang L, Lu F, Suzuki T, Zhang Y
  TITLE     Maternal H3K27me3 controls DNA methylation-independent imprinting.
  JOURNAL   Nature. 2017 Jul 19. doi: 10.1038/nature23262.
  PUBMED    28723896
REFERENCE   2  (bases 1 to 7503)
  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: "Nature. 
            2017 Jul 19. doi: 10.1038/nature23262."
COMMENT     Sequence Label: pcDNA-Flag-Kdm6b(1025-End)_H1390A-pA
FEATURES             Location/Qualifiers
     source          1..7503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             911..934
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3006..3035
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3051..3068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3091..3108)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3097..3321
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3367..3795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3454..3473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3664..3685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3804..3824)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3809..4139
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3990..4125
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4052..4071
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4206..5000
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4260..4279)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4870..4889
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5176..5297
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5213..5232)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5267..5286
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5346..5362)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5346..5362)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5359..5381)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5370..5386
                     /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(5394..5400,5401..5418,5419..5424))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5439..5460
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5577..5594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5748..6336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5828..5847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6507..7298,7299..7367))
                     /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     7130..7149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7368..7472)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttatg gactacaaag acgatgacga caagggatcc gagatccagg gccgggagaa
      961 ggcccggccc gatgtcggtg gggtttccaa agtcaagaca cccacagctc cagcaccccc
     1021 gcctgctcct gcacccgctg ctcagccaac acccccatca gctcctgtcc ctgggaagaa
     1081 gactcgtgag gaggctccgg ggcctccagg tgtgagccgg gcagatatgc tgaagctccg
     1141 gtcacttagt gaggggcctc ccaaggagct gaagatcagg ctcatcaagg tggaaagtgg
     1201 ggacaaggag acctttatcg cctctgaggt ggaagagcgg cggctgcgca tggcagacct
     1261 caccatcagc cactgtgccg ccgatgtcat gcgtgccagc aagaatgcca aggtgaaagg
     1321 gaaattccga gagtcctacc tgtcccctgc ccagtctgtg aaacccaaga tcaacactga
     1381 ggagaagctg ccccgggaaa aactcaaccc ccctaccccc agcatctatt tggagagcaa
     1441 acgagatgcc ttctcgccgg tcctgctaca gttctgtaca gacccccgga accccatcac
     1501 cgtcatcagg ggcctggctg gttcacttcg gctcaactta ggccttttct ccaccaagac
     1561 tctggtggag gcgagcggtg aacatacggt ggaggtccgt acccaagtac agcagccctc
     1621 agacgagaac tgggacctga caggtaccag acaaatctgg ccctgtgaga gctcccgttc
     1681 ccacaccacc atcgctaaat acgcacagta ccaggcctcg tccttccagg agtcactgca
     1741 ggaggagagg gagagtgagg atgaggaatc cgaggaacca gacagcacta caggaacctc
     1801 tcccagcagt gcaccggacc ccaagaacca tcacatcatc aagtttggca ctaacatcga
     1861 cctgtctgat gccaagaggt ggaagccaca gctacaggag ctgctgaaac tgcccgcctt
     1921 catgcgggta acatccacag gcaacatgct cagccacgtg ggccacacca tcctgggcat
     1981 gaacaccgtg cagctataca tgaaggtccc tggcagccga acgccaggcg ctcaagagaa
     2041 taacaatttc tgctcagtca acatcaacat tggccctggg gactgcgagt ggttcgcggt
     2101 acatgagcac tattgggaga ccatcagcgc cttctgcgac cggcatggtg tggactactt
     2161 gactggttcc tggtggccaa tcttggatga cctctatgcg tccaatattc ctgtttaccg
     2221 cttcgtgcag cgccctggag accttgtgtg gattaatgca gggactgtac attgggtgca
     2281 ggctaccggc tggtgcaaca acattgcctg gaacgtgggg cccctcaccg cctatcagta
     2341 ccagctggcc ctggagcgat atgagtggaa cgaggtgaag aacgtcaagt ccattgtgcc
     2401 catgattcat gtgtcctgga acgtcgctcg aacggtcaag atcagcgatc ctgacttgtt
     2461 caagatgatc aagttctgcc tcctgcagtc aatgaagcac tgtcaggtac agcgggagag
     2521 cctggtgcgg gcagggaaga agatcgctta ccaaggccgt gtcaaagacg agcctgccta
     2581 ctactgcaac gaatgcgacg tggaggtgtt caacatcctg ttcgttacaa gtgagaatgg
     2641 cagccgaaac acgtacctgg tgcactgcga gggctgtgcg cgccgtcgca gcgcgggcct
     2701 acagggcgtg gtggtgctag agcagtaccg cacggaggag ctggcgcagg cctacgatgc
     2761 cttcacactg gctcccgcca gcacgtctcg atgagcggcc gctcgactat gggcaccaaa
     2821 gaacctgtaa acgttatctt tttaaattga atgtgcacaa ataaaagttt ggaaaagaaa
     2881 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2941 aaaaaaaaaa aaaaaaaaaa tacgcatcat acgctgtggc gtacctcgag tctagagggc
     3001 ccttcgaaca aaaactcatc tcagaagagg atctgaatat gcataccggt catcatcacc
     3061 atcaccattg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3121 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3181 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3241 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3301 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3361 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3421 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3481 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc
     3541 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3601 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3661 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3721 atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3781 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3841 ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3901 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3961 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4021 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4081 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4141 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     4201 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4261 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4321 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4381 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4441 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4501 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4561 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4621 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4681 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4741 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4801 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4861 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4921 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4981 ttcttgacga gttcttctga gcgggactct ggggttcgcg aaatgaccga ccaagcgacg
     5041 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5101 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5161 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5221 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5281 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5341 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5401 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5461 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5521 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5581 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5641 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5701 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5761 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5821 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5881 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5941 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6001 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6061 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6121 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6181 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6241 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6301 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6361 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6421 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6481 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6541 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6601 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6661 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6721 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6781 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6841 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6901 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6961 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7021 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7081 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7141 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7201 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7261 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7321 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7381 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7441 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7501 gtc
//
