LOCUS       sequence-137202-        6385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6385)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 6385)
  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"
COMMENT     Sequence Label: KDM4D
FEATURES             Location/Qualifiers
     source          1..6385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1246..1263
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1316..1334)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1330..1377
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1414..1869
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1501..1520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1711..1732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1962..2777)
                     /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(1962..2777)
                     /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     2685..2704
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2769..2788
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2899..3487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3388..3407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3641..3658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3673..3815
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3741..3760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3901..3923
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3917..4108)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4917..5999)
                     /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     5961..5980
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6000..6077)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6283..6302
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggaa actatgaagt ctaaggccaa
      181 ctgtgcccag aatccaaatt gtaacataat gatatttcat ccaaccaaag aggagtttaa
      241 tgattttgat aaatatattg cttacatgga atcccaaggt gcacacagag ctggcttggc
      301 taagataatt ccacccaaag aatggaaagc cagagagacc tatgataata tcagtgaaat
      361 cttaatagcc actcccctcc agcaggtggc ctctgggcgg gcaggggtgt ttactcaata
      421 ccataaaaaa aagaaagcca tgactgtggg ggagtatcgc catttggcaa acagtaaaaa
      481 atatcagact ccaccacacc agaatttcga agatttggag cgaaaatact ggaagaaccg
      541 catctataat tcaccgattt atggtgctga catcagtggc tccttgtttg atgaaaacac
      601 taaacaatgg aatcttgggc acctgggaac aattcaggac ctgctggaaa aggaatgtgg
      661 ggttgtcata gaaggcgtca atacacccta cttgtacttt ggcatgtgga aaaccacgtt
      721 tgcttggcat acagaggaca tggaccttta cagcatcaac tacctgcacc ttggggagcc
      781 caaaacttgg tatgtggtgc ccccagaaca tggccagcgc ctggaacgcc tggccaggga
      841 gctcttccca ggcagttccc ggggttgtgg ggccttcctg cggcacaagg tggccctcat
      901 ctcgcctaca gttctcaagg aaaatgggat tcccttcaat cgcataactc aggaggctgg
      961 agagttcatg gtgacctttc cctatggcta ccatgctggc ttcaaccatg gtttcaactg
     1021 cgcagaggcc atcaattttg ccactccgcg atggattgat tatggcaaaa tggcctccca
     1081 gtgtagctgt ggggaggcaa gggtgacctt ttccatggat gccttcgtgc gcatcctgca
     1141 acctgagcgc tatgacctgt ggaaacgtgg gcaagaccgg tgacagtaaa ggtggatacg
     1201 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     1261 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1321 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1381 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1441 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1501 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1561 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1621 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1681 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1741 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1801 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     1861 ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1921 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1981 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2041 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2101 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2161 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2221 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2281 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2341 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2401 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2461 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2521 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2581 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2641 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2701 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2761 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2821 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2881 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2941 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3001 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3061 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3121 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3181 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3241 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3301 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3361 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3421 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3481 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3541 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3601 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3661 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3721 catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3781 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3841 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3901 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3961 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4021 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4081 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4141 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4201 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4261 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4321 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4381 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4441 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4501 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4561 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4621 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4681 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4741 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4801 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4861 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4921 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4981 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5041 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5101 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5161 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5221 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5281 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5341 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5401 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5461 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5521 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5581 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5641 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5701 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5761 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5821 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5881 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5941 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6001 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6061 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6121 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     6181 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6241 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6301 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6361 gatcgagatc tcgatcccgc gaaat
//
