LOCUS       sequence-322541-        6500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  luciferase-based PhoCl screening construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  AUTHORS   Lu X, Wen Y, Zhang S, Zhang W, Chen Y, Shen Y, Lemieux MJ, Campbell 
            RE
  TITLE     Photocleavable proteins that undergo fast and efficient 
            dissociation.
  JOURNAL   Chem Sci. 2021 May 31;12(28):9658-9672. doi: 10.1039/d1sc01059j. 
            eCollection 2021 Jul 21.
  PUBMED    34349937
REFERENCE   2  (bases 1 to 6500)
  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"; doi: 
            "10.1039/d1sc01059j"; journalName: "Chem Sci"; date: "2021-05-31- 
            31"; volume: "12"; issue: "28"; pages: "9658-9672"
COMMENT     Sequence Label: pBad-LgBiT-PhoCl1-SmBiT-MBP
FEATURES             Location/Qualifiers
     source          1..6500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             331..348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             352..384
                     /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(388..396,397..411)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             946..1671
                     /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             1762..2859
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2716..2725
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2833..2856
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(2901..2918)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3071..3157
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3249..3276
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3295..3386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3387..3455,3456..4247)
                     /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(3605..3624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4418..5006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4907..4926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5160..5177
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5192..5332
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5258..5277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5418..5440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5596..6474)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc
      361 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc
      421 tcgaggatgg tcttcacact cgaagatttc gttggggact gggaacagac agccgcgtac
      481 aacctggacc aagtccttga acagggaggt gtgtccagtt tgctgcagaa tctcgcggtg
      541 tccgtaactc cgatccaaag gattgtccgc agcggtgaaa atgcgctgaa gatcgacatc
      601 catgtcatca tcccgtatga aggtctgagc gcggaccaaa tggcacagat cgaagaagtg
      661 tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgcc gtatggcaca
      721 ctggtaatcg acggggttac gccgaacatg ctgaactatt tcggacggcc gtatgaaggc
      781 atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa
      841 attatcgacg agcgcctgat cacccccgac ggctccatgc tgttccgagt aaccatcaac
      901 agcgggagta gcggaggcgg ttctagcggc ggagaacctg gtaccgtgat ccctgactac
      961 ttcaagcaga gcttccccga gggctacagc tgggagcgca gcatgaccta cgaggacggc
     1021 ggcatctgca tcgccaccaa cgacatcaca atggaggggg acagcttcat caacaagatc
     1081 cacttcaagg gcacgaactt cccccccaac ggccccgtga tgcagaagag gaccgtgggc
     1141 tgggaggcca gcaccgagaa gatgtacgag cgcgacggcg tgctgaaggg cgacgtgaag
     1201 atgaagctgc tgctgaaggg cggcggccac tatcgctgcg actaccgcac cacctacaag
     1261 gtcaagcaga agcccgtaaa gctgcccgac taccacttcg tggaccaccg catcgagatc
     1321 ctgagccacg acaaggacta caacaaggtg aagctgtacg agcacgccgt ggcccgcaac
     1381 tccaccgaca gcatggacga gctgtacaag ggtggcagcg gtggcatggt gagcaagggc
     1441 gaggagacca ttacaagcgt gatcaagcct gacatgaaga acaagctgcg catggagggc
     1501 aacgtgaacg gccacgcctt cgtgatcgag ggcgagggca gcggcaagcc cttcgagggc
     1561 atccagacga ttgatttgga ggtgaaggag ggcgccccgc tgcccttcgc ctacgacatc
     1621 ctgaccaccg ccttccacta cggcaaccgc gtgttcacca agtacccacg gtctagaggt
     1681 tcgtccggtg gcggaagcag tggcggcgtg actggataca gactttttga agagatcctg
     1741 gaattcgggg gtggaggttc aaaaatcgaa gaaggtaaac tggtaatctg gattaacggc
     1801 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
     1861 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
     1921 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
     1981 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
     2041 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
     2101 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
     2161 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
     2221 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
     2281 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
     2341 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
     2401 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
     2461 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
     2521 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
     2581 gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     2641 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     2701 gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc
     2761 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     2821 cgtcagactg tcgatgaagc cctgaaagac gcgcagactt aaaagcttgg ctgttttggc
     2881 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata
     2941 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     3001 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     3061 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     3121 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt
     3181 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca
     3241 aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt
     3301 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3361 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3421 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3481 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3541 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3601 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     3661 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3721 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3781 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3841 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3901 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3961 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4021 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4081 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4141 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4201 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4261 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4321 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4381 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4441 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4501 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4561 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4621 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4681 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4741 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4801 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4861 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4921 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4981 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5041 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5101 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5161 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     5221 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     5281 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5341 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5401 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5461 acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cggcatgcat aatgtgcctg
     5521 tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc cgtcaattgt
     5581 ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt cttcacaacc
     5641 ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg agaaatagag
     5701 ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa
     5761 aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa
     5821 ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct
     5881 ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac
     5941 ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa
     6001 ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt
     6061 aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa
     6121 ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg
     6181 aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat
     6241 ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt
     6301 tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg
     6361 tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag
     6421 atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt
     6481 catactcccg ccattcagag
//
