LOCUS       sequence-365741-        7631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human RNA demythelase protein FTO 32-505 amino acid 
            sequence with N-terminal fusion with the N-domain of EI ( e.coli 
            protein).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7631)
  AUTHORS   Khatiwada B, Purslow JA, Underbakke ES, Venditti V
  TITLE     N-terminal fusion of the N-terminal domain of bacterial enzyme I 
            facilitates recombinant expression and purification of the human RNA
            demethylases FTO and Alkbh5.
  JOURNAL   Protein Expr Purif. 2019 Nov 15;167:105540. doi: 
            10.1016/j.pep.2019.105540.
  PUBMED    31740367
REFERENCE   2  (bases 1 to 7631)
  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: "Protein 
            Expr Purif."; date: "2019-11-15"; volume: "167:105540. doi"; pages: 
            " 10.1016/j.pep.2019.105540"
COMMENT     Sequence Label: pET-EIN-FTO(32-505)
FEATURES             Location/Qualifiers
     source          1..7631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     930..949
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(969..1046)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1252..1271
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1355..1374
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1355..1373
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1374..1398
                     /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             1413..1435
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1454..1471
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2231..2251
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3717..3734
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3787..3805)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3801..3848
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3885..4340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3972..3991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4182..4203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4366..4470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4471..4539,4540..5331)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4689..4708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5502..6090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5991..6010
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6244..6261
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6276..6415
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6341..6360)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6501..6523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6517..6708)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7517..7631,1..968))
                     /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"
ORIGIN
        1 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
       61 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
      121 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
      181 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
      241 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
      301 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
      361 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
      421 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
      481 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
      541 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      601 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      661 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
      721 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
      781 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
      841 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
      901 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
      961 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     1021 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     1081 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     1141 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     1201 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     1261 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     1321 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1381 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1441 catgggcagc agccatcatc atcatcacca tagcagcatt tcaggcattt tagcatcccc
     1501 gggtatcgct ttcggtaaag ctctgcttct gaaagaagac gaaattgtca ttgaccggaa
     1561 aaaaatttct gccgaccagg ttgatcagga agttgaacgt tttctgagcg gtcgtgccaa
     1621 ggcatcagcc cagctggaaa cgatcaaaac gaaagctggt gaaacgttcg gtgaagaaaa
     1681 agaagccatc tttgaagggc atattatgct gctcgaagat gaggagctgg agcaggaaat
     1741 catagccctg attaaagata agcacatgac agctgacgca gctgctcatg aagttatcga
     1801 aggtcaggct tctgccctgg aagagctgga tgatgaatac ctgaaagaac gtgcggctga
     1861 cgtacgtgat atcggtaagc gcctgctgcg caacatcctg ggcctgaaga ttatcgacct
     1921 gagcgccatt caggatgaag tcattctggt tgccgctgac ctgacgccgt ccgaaaccgc
     1981 acagctgaac ctgaagaagg tgctgggttt catcaccgac gcgggtggcc gtacttccca
     2041 cacctctatc atggcgcgtt ctctggaact acctgctatc gtgggtaccg gtagcgtcac
     2101 ctctcaggtg aaaaatgacg actatctgat tctggatgcc gtaaataatc aggtttacgt
     2161 caatccaacc aacgaagtta ttgataaaat gcgcgctgtt caggagcaag tggcttctga
     2221 acatagcagc gaaaatctgt attttcagag caccccgaaa gacgacgaat tttacaagca
     2281 atggcaactg aagtacccga aactgatcct gcgtgaagca tcgagcgtga gcgaagaact
     2341 gcataaagaa gttcaagaag catttctgac cctgcataag cacggctgcc tgttccgtga
     2401 tctggtgcgc attcagggta aagacctgct gaccccggtt agccgtattc tgatcggcaa
     2461 cccgggttgt acgtataagt acctgaatac ccgcctgttt acggtcccgt ggccggtgaa
     2521 aggctctaac attaagcaca ccgaagcgga aatcgcggcc gcatgcgaaa cgttcctgaa
     2581 actgaatgat tatctgcaga ttgaaaccat ccaagctctg gaagaactgg ctgcgaaaga
     2641 aaaggctaac gaagatgcgg tcccgctgtg tatgtcagcg gactttccgc gtgtgggcat
     2701 gggtagctct tataatggcc aagatgaagt tgacatcaaa tcgcgcgccg catacaacgt
     2761 cacgctgctg aattttatgg acccgcagaa aatgccgtat ctgaaggaag aaccgtactt
     2821 cggcatgggt aaaatggcgg tgtcatggca tcacgatgaa aacctggttg accgttcggc
     2881 tgtggcggtt tattcatact cgtgcgaagg tccggaagaa gaaagtgaag atgactccca
     2941 tctggaaggc cgcgatccgg acatttggca cgtgggtttt aaaattagct gggatattga
     3001 aaccccgggt ctggcaatcc cgctgcatca gggtgactgc tattttatgc tggatgacct
     3061 gaatgccacc catcagcact gtgttctggc aggcagccaa ccgcgtttca gttccacgca
     3121 ccgcgtcgcc gaatgttcta ccggtacgct ggattacatt ctgcagcgtt gccaactggc
     3181 actgcagaac gtttgtgatg acgtcgataa tgatgacgtc agcctgaaat cttttgaacc
     3241 ggccgtgctg aagcagggcg aagaaatcca taacgaagtt gaattcgaat ggctgcgcca
     3301 gttttggttc caaggtaatc gttatcgcaa atgcaccgat tggtggtgtc agccgatggc
     3361 ccaactggaa gcactgtgga aaaagatgga aggcgtgacg aacgcagttc tgcatgaagt
     3421 caaacgtgaa ggtctgccgg tggaacagcg caacgaaatt ctgaccgcca tcctggcaag
     3481 tctgacggct cgtcagaatc tgcgtcgcga atggcacgcg cgctgccaat cccgtattgc
     3541 tcgcaccctg ccggcggatc agaaaccgga atgtcgtccg tactgggaaa aggatgacgc
     3601 cagcatgccg ctgccgtttg acctgaccga catcgtctct gaactgcgcg gccaactgct
     3661 ggaagcgaaa ccgtaagaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     3721 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3781 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgagga
     3841 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3901 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3961 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4021 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4081 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4141 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4201 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4261 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4321 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4441 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4501 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4561 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4621 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4681 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4741 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4801 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4861 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4921 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4981 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     5041 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5101 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5161 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5221 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5281 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5341 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5401 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5461 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5521 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5581 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5641 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5701 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5761 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5821 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5881 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5941 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6001 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6061 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6121 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6181 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6241 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     6301 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     6361 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     6421 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6481 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6541 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     6601 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     6661 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     6721 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     6781 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     6841 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     6901 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     6961 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     7021 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     7081 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     7141 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     7201 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     7261 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     7321 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     7381 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     7441 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     7501 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7561 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     7621 ttcttttcac c
//
