LOCUS       sequence-72771-e        5866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human Kdm2b CxxC-PHD domain with N-His6-TEV 
            and C-Avi tags. For use in BioCAP non-methylated DNA purification. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5866)
  TITLE     Klose Lab BioCAP plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5866)
  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            "Unpublished"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: pNIC28-hKdm2b-CxxC-PHD
FEATURES             Location/Qualifiers
     source          1..5866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             570..614
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             680..697
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(750..768)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      764..811
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      848..1303
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(935..954)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1145..1166
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1396..2211)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1396..2211)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2119..2138
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2203..2222
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2333..2921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2822..2841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3075..3092
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3107..3249
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3175..3194)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3335..3357
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3351..3542)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4351..5433)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5395..5414
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5434..5511)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5717..5736
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5820..5839
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5820..5838
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5839..5863
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg aaccggacaa
      121 cggcaggagc tcggcggcgc cggacgcgat gccgcaagtg cgaggcctgc ctgcggaccg
      181 agtgcggaga gtgccacttc tgcaaggaca tgaagaagtt cgggggcccc gggcgcatga
      241 agcagagctg catcatgcgg cagtgcatcg cgccagtgct gccccacacc gccgtgtgcc
      301 ttgtgtgtgg cgaggcgggg aaggaagaca cggtggaaga ggaggaaggc aagtttaacc
      361 tcatgctcat ggagtgctcc atctgcaatg aaatcatcca ccctggatgc cttaagatta
      421 aggagtcaga gggtgtggtc aacgacgagc ttccaaactg ctgggagtgt ccgaagtgta
      481 accacgccgg caagaccggg aaacaaaagc gtggccctgg ctttaagtac gcctccaacc
      541 tgcccggctc cctgctcaag gagggtggcg gtctgaacga catcttcgag gctcagaaaa
      601 tcgaatggca cgaataacag taaaggtgga tacggatccg aattcgagct ccgtcgacaa
      661 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      721 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      781 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
      841 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      901 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      961 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1021 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1081 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1141 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1201 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1261 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg
     1321 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1381 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1441 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1501 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1561 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1621 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1681 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1741 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     1801 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1861 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     1921 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     1981 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2041 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2101 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2161 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     2221 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2281 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2341 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2401 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2461 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2521 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2581 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2641 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2701 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2761 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2821 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2881 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2941 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3001 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3061 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3121 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     3181 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3241 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3301 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3361 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3421 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3481 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3541 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3601 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3661 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3721 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3781 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3841 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3901 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3961 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4021 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4081 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4141 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4201 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4261 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4321 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4441 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4501 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4561 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4621 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4681 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4741 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4801 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4861 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4921 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4981 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5041 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5101 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5161 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5221 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5281 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5341 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5401 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5461 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5521 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5581 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5641 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5701 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5761 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     5821 aatacgactc actatagggg aattgtgagc ggataacaat tcccct
//
