LOCUS       sequence-193858-        7502 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 7502)
  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 7502)
  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..7502
                     /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..4138
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3989..4124
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4051..4070
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4205..4999
                     /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(4259..4278)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4869..4888
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5175..5296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5212..5231)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5266..5285
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5345..5361)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5345..5361)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5358..5380)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5369..5385
                     /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(5393..5399,5400..5417,5418..5423))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5438..5459
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5576..5593)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5747..6335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5827..5846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6506..7297,7298..7366))
                     /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     7129..7148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7367..7471)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ggggctccct ttagggttcc
     3541 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3601 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3661 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3721 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3781 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3841 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3901 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3961 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4021 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4081 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4141 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     4201 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4261 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4321 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4381 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4441 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4501 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4561 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4621 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4681 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4741 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4801 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4861 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4921 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4981 tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc
     5041 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5101 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5161 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5221 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5281 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5341 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5401 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5461 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5521 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5581 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5641 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5701 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5761 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5821 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5881 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5941 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6001 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6061 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6121 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6181 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6241 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6301 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6361 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6421 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6481 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6541 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6601 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6661 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6721 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6781 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6841 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6901 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6961 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7021 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7081 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7141 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7201 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7261 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7321 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7381 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7441 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7501 tc
//
