LOCUS       sequence-214326-        5846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RAMA domain from murine MPND.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5846)
  AUTHORS   Kweon SM, Chen Y, Moon E, Kvederaviciute K, Klimasauskas S, Feldman 
            DE
  TITLE     An Adversarial DNA N(6)-Methyladenine-Sensor Network Preserves 
            Polycomb Silencing.
  JOURNAL   Mol Cell. 2019 Apr 5. pii: S1097-2765(19)30222-9. doi: 
            10.1016/j.molcel.2019.03.018.
  PUBMED    30982744
REFERENCE   2  (bases 1 to 5846)
  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: "Mol Cell. 
            2019 Apr 5. pii: S1097-2765(19)30222-9. doi: 
            10.1016/j.molcel.2019.03.018."
COMMENT     Sequence Label: pET30a-RAMA
FEATURES             Location/Qualifiers
     source          1..5846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      21..609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     510..529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     763..780
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    795..934
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(860..879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1020..1042
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1036..1227)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2036..3118)
                     /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     3080..3099
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3119..3196)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3402..3421
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3509..3528
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3509..3527
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3528..3552
                     /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             3567..3589
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3600..3617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3627..3644
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3651..3695
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4182..4205
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4215..4232
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4285..4303)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4299..4346
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4383..4838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4470..4489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4680..4701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4930..5745)
                     /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(4930..5745)
                     /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     5653..5672
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5737..5756
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
       61 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
      121 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
      181 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
      241 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
      301 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
      361 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
      421 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
      481 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
      541 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
      601 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
      661 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
      721 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      781 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      841 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
      901 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
      961 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     1021 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     1081 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     1141 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     1201 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     1261 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     1321 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     1381 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     1441 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     1501 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     1561 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     1621 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     1681 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     1741 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     1801 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     1861 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     1921 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     1981 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     2041 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2101 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     2161 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     2221 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     2281 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     2341 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     2401 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     2461 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     2521 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     2581 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     2641 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     2701 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     2761 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     2821 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     2881 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     2941 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     3001 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     3061 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     3121 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     3181 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     3241 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     3301 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     3361 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     3421 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     3481 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     3541 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
     3601 accatcatca tcatcattct tctggtctgg tgccacgcgg ttctggtatg aaagaaaccg
     3661 ctgctgctaa attcgaacgc cagcacatgg acagcccaga tctggcagct cccgagtctc
     3721 tgtctcccgg ggccacggcc gaggaggcgc cggaggagga tgaggacgac gcggaggccg
     3781 aggaccccga gcgcgggacg ggtagcggag ggcgcagcgg cagcctcggt ggcagcggag
     3841 gcggcacggc agggcctggg atggccctag ggggcgcgct cacgaggcgc gcggtcacgc
     3901 ttcgggtgct gctcaaagac gagctgctgg agcccggtga gggggtgcta tccatctact
     3961 acctgggcag gaagtttact ggggacctgc agctggatgg caggatcgtg tggcaggaaa
     4021 ctggacaagt cttcaactca cccagcgcct gggccacaca ctgcaagaag ctcgtcaacc
     4081 cagccaagaa atctggctgt ggctgggcct ctgtcaagta caagggccag aagctggaca
     4141 aatacaaggc cgcctggctt cgccggcacc agctacatat ggattacaag gatgacgatg
     4201 acaagtgact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     4261 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     4321 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     4381 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4441 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4501 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4561 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4621 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4681 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4741 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4801 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     4861 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4921 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     4981 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     5041 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     5101 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     5161 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     5221 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     5281 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     5341 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     5401 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     5461 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     5521 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     5581 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     5641 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     5701 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     5761 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     5821 cgttccactg agcgtcagac cccgta
//
