LOCUS       sequence-177058-        5975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express PhoCl-MBP in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5975)
  AUTHORS   Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu 
            S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell
            RE
  TITLE     Optogenetic control with a photocleavable protein, PhoCl.
  JOURNAL   Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222.
  PUBMED    28288123
REFERENCE   2  (bases 1 to 5975)
  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: "Nat 
            Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222."
COMMENT     Sequence Label: pBAD/HisB-PhoCl-MBP
FEATURES             Location/Qualifiers
     source          1..5975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..293
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     216..235
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             339..356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             360..392
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(396..404,405..419)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             435..1160
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTY
                     KVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     CDS             1176..2276
                     /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      2133..2142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2250..2273
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2325..2336
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2384..2401)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2554..2640
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2732..2759
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2778..2869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2870..2938,2939..3730)
                     /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(3088..3107)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3901..4489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4390..4409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4643..4660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4675..4815
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4741..4760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4901..4923
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5079..5957)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta
       61 ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag
      121 cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt
      181 ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag
      241 attagcggat cctacctgac gctttttatc gcaactctct actgtttctc catacccgtt
      301 ttttgggcta acaggaggaa ttaaccatgg ggggttctca tcatcatcat catcatggta
      361 tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg
      421 atccgagctc gagtgtgatc cctgactact tcaagcagag cttccccgag ggctacagct
      481 gggagcgcag catgacctac gaggacggcg gcatctgcat cgccaccaac gacatcacaa
      541 tggaggggga cagcttcatc aacaagatcc acttcaaggg cacgaacttc ccccccaacg
      601 gccccgtgat gcagaagagg accgtgggct gggaggccag caccgagaag atgtacgagc
      661 gcgacggcgt gctgaagggc gacgtgaaga tgaagctgct gctgaagggc ggcggccact
      721 atcgctgcga ctaccgcacc acctacaagg tcaagcagaa gcccgtaaag ctgcccgact
      781 accacttcgt ggaccaccgc atcgagatcc tgagccacga caaggactac aacaaggtga
      841 agctgtacga gcacgccgtg gcccgcaact ccaccgacag catggacgag ctgtacaagg
      901 gtggcagcgg tggcatggtg agcaagggcg aggagaccat tacaagcgtg atcaagcctg
      961 acatgaagaa caagctgcgc atggagggca acgtgaacgg ccacgccttc gtgatcgagg
     1021 gcgagggcag cggcaagccc ttcgagggca tccagacgat tgatttggag gtgaaggagg
     1081 gcgccccgct gcccttcgcc tacgacatcc tgaccaccgc cttccactac ggcaaccgcg
     1141 tgttcaccaa gtacccacgg ggaggtggag gtaccatgaa aatcgaagaa ggtaaactgg
     1201 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
     1261 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
     1321 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
     1381 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
     1441 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
     1501 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
     1561 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
     1621 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
     1681 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
     1741 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
     1801 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
     1861 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
     1921 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     1981 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     2041 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     2101 aggaagagtt ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg
     2161 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     2221 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt
     2281 cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag ggaaggattt
     2341 cataaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     2401 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     2461 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     2521 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     2581 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2641 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     2701 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     2761 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2821 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2881 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2941 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3001 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3061 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     3121 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3181 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3241 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3301 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3361 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3421 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3481 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3541 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3601 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3661 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3721 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3781 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3841 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3901 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3961 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4021 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4081 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4141 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4201 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4261 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4321 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4381 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4441 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4501 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4561 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4621 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     4741 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4801 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4861 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4921 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     4981 aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc
     5041 tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca
     5101 tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt
     5161 ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg
     5221 ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc
     5281 cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac
     5341 aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg
     5401 atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc
     5461 gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag
     5521 cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg
     5581 tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat
     5641 tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc
     5701 ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg
     5761 caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga
     5821 atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca
     5881 atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca
     5941 ttttgcgctt cagccatact tttcatactc ccgcc
//
