LOCUS       sequence-231768-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mCherry-tagged BcLOV4 ""dark"" mutant with C292A mutation 
            for expression in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6756)
  AUTHORS   Glantz ST, Berlew EE, Chow BY
  TITLE     Synthetic cell-like membrane interfaces for probing dynamic 
            protein-lipid interactions.
  JOURNAL   Methods Enzymol. 2019;622:249-270. doi: 10.1016/bs.mie.2019.02.015. 
            Epub 2019 Mar 23.
  PUBMED    31155055
REFERENCE   2  (bases 1 to 6756)
  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: "Methods 
            Enzymol."; date: "2019"; volume: "622:249-270. doi"; pages: " 
            10.1016/bs.mie.2019.02.015. Epub 2019 Mar 23"
COMMENT     Sequence Label: His6_BcLOV4_C292A_mCherry_BamUK
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..74
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     158..177
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        158..176
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    177..201
                     /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             216..238
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             255..272
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2088..2798
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2233..2251)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2501..2520
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2809..2826
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2879..2897)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2893..2940
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2977..3432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3064..3083)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3274..3295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3547..4362)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3547..4362)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4270..4289
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4354..4373
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4515..5103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5004..5023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5257..5274
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5445..6527)
                     /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     6489..6508
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6528..6605)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
       61 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
      121 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
      181 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
      241 tacatatggg atcccaccat catcaccacc atgccatggc cacggacgca attgaccact
      301 actatcagaa cagggatgga ccaccgtcga gatccttcag cgatgagtac gattcataca
      361 acaaaaccgc ccctttgcct ccactatcag catgtcctga aagagcccag actccaaatt
      421 caaagcgtat tccgataacc cccaattctg ccaaatctac atctgagagc ccacgccgca
      481 tcactttttc tagggatgcc ggaagtagca caaaaggctc gacacaaaca agtcctagta
      541 accaaggaag tgtaagcagt aggaatggta gcaatggaaa tggtttaggg ctgagtagcg
      601 cgggaaactt tgcagatttc tttagctctg aagtgtttta tattgtgctt cataatccaa
      661 cgaccgcgca tcgattcttg aagttttgtc aaagtcgtgc ttgttcggaa aaccttgaat
      721 ttttgcaaaa gattgatgag tataatcgat taatagacga agcgacacgt cttctttcca
      781 cgatttactc aacctatgtt tcgtcagaag caccaaagca aatcaatatt tccaattctc
      841 atgctcgacg attatcacat gatatcaagg gtgcgacgca taatgttctt cccggccttg
      901 aagacatctt caatgggtcc caggagcaaa ttgagaaatt gctagctagt gatttgtatc
      961 cgcgttttgt aaaacaccag gttactgcct ccgcgactat ggcgttggca aatgataagg
     1021 aaagatatcc aggtctaggt gactgttttt gcttgactga tccaaggcga gccgataacc
     1081 caattgtatt tgcttcggac gggttcgtct ctgtcacggg atattcccga tcagatataa
     1141 tacctcgaaa cgctcgtttc ctacaaggat cgtttaccga tcgacaagca accaaacgtc
     1201 tacgaacttc cattgaaaac cgtgaagaga cagttgaatt acttctgaat tatcgcaaaa
     1261 atggcgatcc attctggaat ctcctttacg tatctccttt acttgacggt aatggggacg
     1321 tcagattctt tctcggtgga caaatcaatt gcagtacgac tattcacagt cgcaccgatg
     1381 ttcttcgaat tttgagtctg aacgatgagg acactgggag ctttatagag gggagtaaca
     1441 agaccccgag tgttcgaagc aaggagtcca ataatgcaag caactatggg aaaacctcat
     1501 tcttcaagag cttcaaaaag tacaaggcga atactgtaga aatccgagac gaagctggga
     1561 tggaaggcga attgatcgat cggatcggaa agctacaatt caaaacacaa gttgaagagt
     1621 tctatacggc ttattcaaag tatcttgtcc tatcttacca accacagtca caaaatctga
     1681 ttgtcaaata cttctctccg ggtatagtgg acatgcttaa tctaaacatg cccaatggcc
     1741 tcgtagctcc tattgtcgat aaagatatct tcaaagttct cacagaacac tccccgtcgt
     1801 ctgtaccgcg caccttcaaa aatgtggtta gagaaggtat caaagccgga cgagcagtat
     1861 cagtcgaaac gggacttctg actggaattg aagagatcaa gaagtcacaa aatattttta
     1921 gtggcacaac ggcaaataga gagagagggt ggaagaaggc cgaggaaaaa tatgtctgtc
     1981 attttacacc ttgtaaagat gagatgggca gagttggatg ggtcgttttg actgtggcgc
     2041 cgaaactgga gagatttgaa ttcggtggtg gatcaggtgg aggatctatg gtgagcaagg
     2101 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     2161 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     2221 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     2281 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2341 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2401 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2461 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2521 ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2581 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2641 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2701 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2761 gccactccac cggcggcatg gacgagctgt acaagtaatg aactcgagca ccaccaccac
     2821 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     2881 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2941 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     3001 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3061 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3121 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3181 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3241 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3301 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3361 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3421 cgcttacaat ttaggtggca cttttcgggg tacccaggtg gcacttttcg gggaaatgtg
     3481 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3541 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3601 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3661 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3721 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3781 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3841 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3901 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     3961 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4021 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4081 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4141 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4201 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4261 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4321 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4381 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gacctaggtc
     4441 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4501 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4561 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4621 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4681 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4741 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4801 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4861 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4921 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4981 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5041 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5101 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5161 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5221 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5281 gaagagcgcc ataatccatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5341 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5401 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5521 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5581 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5641 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5701 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     5761 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5821 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5881 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5941 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6001 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6061 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6121 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6181 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6241 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6301 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6361 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6421 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6481 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6541 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6601 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6661 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     6721 caacagtccc ccggccacgg ggcctgccac catacc
//
