LOCUS       sequence-371437-        6283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FTO in bacterial cells, with two purification tags: His 
            and Twin-Streptag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6283)
  TITLE     Bacterial expression plasmid for production of recombinant FTO 
            protein
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6283)
  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            "Unpublished"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: pPROEX-TwStrp-FTO
FEATURES             Location/Qualifiers
     source          1..6283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    230..246
                     /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)."
     primer_bind     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             279..296
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(306..311,312..335,336..371,372..395)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(1973..1990)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2143..2229
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2321..2348
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2367..2458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2459..2527,2528..3319)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2677..2696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3490..4078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3979..3998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      complement(4326..4754)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4436..4457)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4648..4667
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4792..4811)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4984..5006
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5084..5161
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5162..6244
                     /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     complement(5181..5200)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gtccgtataa tctgtggaat tgtgagcgga
      241 taacaatttc acacaggacc tctagaatgg gtagctcgca tcatcatcat catcattcca
      301 gcggtagcgc ttggagccac ccgcagttcg aaaaaggtgg aggttctggc ggtggatcgg
      361 gaggttcagc gtggagccac ccgcagttcg agaaaggatc caagcgcacc ccgactgccg
      421 aggaacgaga gcgcgaagct aagaaactga ggcttcttga agagcttgaa gacacttggc
      481 tcccttatct gacccccaaa gatgatgaat tctatcagca gtggcagctg aaatatccta
      541 aactaattct ccgagaagcc agcagtgtat ctgaggagct ccataaagag gttcaagaag
      601 cctttctcac actgcacaag catggctgct tatttcggga cctggttagg atccaaggca
      661 aagatctgct cactccggta tctcgcatcc tcattggtaa tccaggctgc acctacaagt
      721 acctgaacac caggctcttt acggtcccct ggccagtgaa agggtctaat ataaaacaca
      781 ccgaggctga aatagccgct gcttgtgaga ccttcctcaa gctcaatgac tacctgcaga
      841 tagaaaccat ccaggctttg gaagaacttg ctgccaaaga gaaggctaat gaggatgctg
      901 tgccattgtg tatgtctgca gatttcccca gggttgggat gggttcatcc tacaacggac
      961 aagatgaagt ggacattaag agcagagcag catacaacgt aactttgctg aatttcatgg
     1021 atcctcagaa aatgccatac ctgaaagagg aaccttattt tggcatgggg aaaatggcag
     1081 tgagctggca tcatgatgaa aatctggtgg acaggtcagc ggtggcagtg tacagttata
     1141 gctgtgaagg ccctgaagag gaaagtgagg atgactctca tctcgaaggc agggatcctg
     1201 atatttggca tgttggtttt aagatctcat gggacataga gacacctggt ttggcgatac
     1261 cccttcacca aggagactgc tatttcatgc ttgatgatct caatgccacc caccaacact
     1321 gtgttttggc cggttcacaa cctcggttta gttccaccca ccgagtggca gagtgctcaa
     1381 caggaacctt ggattatatt ttacaacgct gtcagttggc tctgcagaat gtctgtgacg
     1441 atgtggacaa tgatgatgtc tctttgaaat cctttgagcc tgcagttttg aaacaaggag
     1501 aagaaattca taatgaggtc gagtttgagt ggctgaggca gttttggttt caaggcaatc
     1561 gatacagaaa gtgcactgac tggtggtgtc aacccatggc tcaactggaa gcactgtgga
     1621 agaagatgga gggtgtgaca aatgctgtgc ttcatgaagt taaaagagag gggctccccg
     1681 tggaacaaag gaatgaaatc ttgactgcca tccttgcctc gctcactgca cgccagaacc
     1741 tgaggagaga atggcatgcc aggtgccagt cacgaattgc ccgaacatta cctgctgatc
     1801 agaagccaga atgtcggcca tactgggaaa aggatgatgc ttcgatgcct ctgccgtttg
     1861 acctcacaga catcgtttca gaactcagag gtcagcttct ggaagcaaaa ccctaactcg
     1921 aggcatgcgg taccaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     1981 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     2041 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     2101 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     2161 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2221 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     2281 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     2341 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     2401 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2461 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2521 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2581 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2641 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2701 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2761 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2821 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2881 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2941 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3001 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3061 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3121 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3181 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3241 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3301 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3361 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3421 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3481 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3541 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3601 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3661 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3721 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3781 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3841 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3901 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3961 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4021 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4081 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4141 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4201 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4261 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcataatttt
     4321 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4381 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     4441 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     4501 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     4561 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     4621 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     4681 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     4741 gctacagggc gcgtcccatt cgccattcag gctgctatgg tgcactctca gtacaatctg
     4801 ctctgatgcc gcatagttaa gccagtacca gtcacgtagc gatatcgcta cgtgactggt
     4861 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4921 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4981 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5041 agatcaattc gcgcgcgaag gcgaagcggc atgcatttac gttgacacca tcgaatggtg
     5101 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     5161 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     5221 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     5281 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     5341 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     5401 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     5461 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     5521 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     5581 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     5641 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     5701 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     5761 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     5821 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     5881 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     5941 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     6001 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     6061 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     6121 cgtctcactg gtgaaaagaa aaaccaccct ggcacccaat acgcaaaccg cctctccccg
     6181 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6241 gtgagcgcaa cgcaattaat gtaagttagc gcgaattgat ctg
//
