LOCUS       sequence-238568-        6585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-EX-HA-FRB-MBD in bacteria for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6585)
  AUTHORS   Han Y, Branon TC, Martell JD, Boassa D, Shechner D, Ellisman MH, 
            Ting A
  TITLE     Directed Evolution of Split APEX2 Peroxidase.
  JOURNAL   ACS Chem Biol. 2019 Mar 8. doi: 10.1021/acschembio.8b00919.
  PUBMED    30848125
REFERENCE   2  (bases 1 to 6585)
  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: "ACS Chem 
            Biol. 2019 Mar 8. doi: 10.1021/acschembio.8b00919."
COMMENT     Sequence Label: His6x-EX-HA-FRB-MBD_pYFJ16
FEATURES             Location/Qualifiers
     source          1..6585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     CDS             109..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             679..1779
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1636..1645
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1753..1776
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1828..1839
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      2042..2136
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2180..2839
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2246..2265)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2904..2990
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3083..4165)
                     /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     4127..4146
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4166..4243)
                     /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."
     primer_bind     complement(4327..4349)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4435..4575
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4490..4509
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4590..4607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4761..5349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4841..4860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5520..6311,6312..6380))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6143..6162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6381..6485)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6553..6571
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcaaattaa ctatgaaaca tcaccatcac
      121 catcaccata tggggctgct gcagctgccc agcgacaaag ccctgctgtc cgatcccgtg
      181 ttcagacctc tggtcgataa gtatgcagcc gacgaggatg cttttttcgc agattacgca
      241 gaagcacatc agaagctgtc agaactggga tttgccgacg ccggctcggg cgggagtacc
      301 ggttacccat acgacgttcc agactacggt agtgggagcg gtgtggccat cctgtggcac
      361 gagatgtggc atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg
      421 aaaggcatgt ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact
      481 ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg
      541 tgcaggaagt acatgaaatc agggaatgtc aaggacctca cccaagcctg ggacctctat
      601 tatcatgtgt tccgacgaat ctcaagtgct ggtggtagtg ctggtggtag tgctggtggt
      661 agtgctggtg gtagtgctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc
      721 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
      781 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
      841 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
      901 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
      961 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
     1021 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
     1081 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
     1141 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
     1201 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
     1261 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
     1321 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
     1381 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
     1441 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
     1501 gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     1561 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     1621 gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc
     1681 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     1741 cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac
     1801 aacaataaca ataacaacaa cctcgggatc gagggaaggg gataataagg cgcgccttac
     1861 ccgcccatgc gggcaacttt ctcttcgatt tgccggattt tttccggcat ttcttgcgca
     1921 atgtggcgaa acgcctcgct gatacgcgga tctctggcga ccgtttgcca gaactgctgc
     1981 gccagcggga tcgccgttgg ggatccgtcg acctgcagcc aagcttaatt agctgagctt
     2041 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     2101 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagcttgg cgagattttc
     2161 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     2221 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     2281 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     2341 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaatttcg
     2401 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     2461 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     2521 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     2581 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     2641 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     2701 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     2761 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     2821 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggggt
     2881 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     2941 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccctcta
     3001 gattacgtgc agtcgatgat aagctgtcaa acatgagaat tgtgcctaat gagtgagcta
     3061 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     3181 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     3241 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     3301 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     3361 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     3421 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     3481 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3541 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     3601 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     3661 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     3721 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     3781 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     3841 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     3901 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     3961 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     4021 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     4081 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     4141 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     4201 atcatgccat accgcgaaag gttttgcacc attcgatggt ttcggaattt cgggcagcgt
     4261 tgggtcctgg ccacgggtgc gcatgatcta gagctgcctc gcgcgtttcg gtgatgacgg
     4321 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4381 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4441 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4501 cagattgtac tgagagtgca ccacatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4561 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4621 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4681 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4741 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4801 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4861 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4921 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4981 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5041 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5101 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5161 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5221 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5281 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5341 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5401 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5461 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5521 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5581 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5641 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5701 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5761 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5821 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5881 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5941 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6001 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6061 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6481 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6541 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcac
//
