LOCUS       sequence-231770-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mCherry-tagged BcLOV4 permanently-""lit"" mutant with Q355N 
            mutation for expression in bacterial cell.
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_Q355N_mCherry_BamUK
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     156..175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        156..174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    175..199
                     /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             214..236
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             253..270
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2086..2796
                     /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(2231..2249)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2499..2518
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2807..2824
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2877..2895)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2891..2938
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2975..3430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3062..3081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3272..3293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3545..4360)
                     /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(3545..4360)
                     /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     4268..4287
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4352..4371
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4513..5101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5002..5021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5255..5272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5443..6525)
                     /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     6487..6506
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6526..6603)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
       61 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
      121 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      181 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      241 catatgggat cccaccatca tcaccaccat gccatggcca cggacgcaat tgaccactac
      301 tatcagaaca gggatggacc accgtcgaga tccttcagcg atgagtacga ttcatacaac
      361 aaaaccgccc ctttgcctcc actatcagca tgtcctgaaa gagcccagac tccaaattca
      421 aagcgtattc cgataacccc caattctgcc aaatctacat ctgagagccc acgccgcatc
      481 actttttcta gggatgccgg aagtagcaca aaaggctcga cacaaacaag tcctagtaac
      541 caaggaagtg taagcagtag gaatggtagc aatggaaatg gtttagggct gagtagcgcg
      601 ggaaactttg cagatttctt tagctctgaa gtgttttata ttgtgcttca taatccaacg
      661 accgcgcatc gattcttgaa gttttgtcaa agtcgtgctt gttcggaaaa ccttgaattt
      721 ttgcaaaaga ttgatgagta taatcgatta atagacgaag cgacacgtct tctttccacg
      781 atttactcaa cctatgtttc gtcagaagca ccaaagcaaa tcaatatttc caattctcat
      841 gctcgacgat tatcacatga tatcaagggt gcgacgcata atgttcttcc cggccttgaa
      901 gacatcttca atgggtccca ggagcaaatt gagaaattgc tagctagtga tttgtatccg
      961 cgttttgtaa aacaccaggt tactgcctcc gcgactatgg cgttggcaaa tgataaggaa
     1021 agatatccag gtctaggtga ctgtttttgc ttgactgatc caaggcgagc cgataaccca
     1081 attgtatttg cttcggacgg gttcgtctct gtcacgggat attcccgatc agatataata
     1141 cctcgaaact gtcgtttcct acaaggatcg tttaccgatc gacaagcaac caaacgtcta
     1201 cgaacttcca ttgaaaaccg tgaagagaca gttgaattac ttctgaatta tcgcaaaaat
     1261 ggcgatccat tctggaatct cctttacgta tctcctttac ttgacggtaa tggggacgtc
     1321 agattctttc tcggtggaaa catcaattgc agtacgacta ttcacagtcg caccgatgtt
     1381 cttcgaattt tgagtctgaa cgatgaggac actgggagct ttatagaggg gagtaacaag
     1441 accccgagtg ttcgaagcaa ggagtccaat aatgcaagca actatgggaa aacctcattc
     1501 ttcaagagct tcaaaaagta caaggcgaat actgtagaaa tccgagacga agctgggatg
     1561 gaaggcgaat tgatcgatcg gatcggaaag ctacaattca aaacacaagt tgaagagttc
     1621 tatacggctt attcaaagta tcttgtccta tcttaccaac cacagtcaca aaatctgatt
     1681 gtcaaatact tctctccggg tatagtggac atgcttaatc taaacatgcc caatggcctc
     1741 gtagctccta ttgtcgataa agatatcttc aaagttctca cagaacactc cccgtcgtct
     1801 gtaccgcgca ccttcaaaaa tgtggttaga gaaggtatca aagccggacg agcagtatca
     1861 gtcgaaacgg gacttctgac tggaattgaa gagatcaaga agtcacaaaa tatttttagt
     1921 ggcacaacgg caaatagaga gagagggtgg aagaaggccg aggaaaaata tgtctgtcat
     1981 tttacacctt gtaaagatga gatgggcaga gttggatggg tcgttttgac tgtggcgccg
     2041 aaactggaga gatttgaatt cggtggtgga tcaggtggag gatctatggt gagcaagggc
     2101 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     2161 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2221 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2281 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2341 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2401 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2461 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagact
     2521 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     2581 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     2641 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     2701 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     2761 cactccaccg gcggcatgga cgagctgtac aagtaatgaa ctcgagcacc accaccacca
     2821 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2881 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2941 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3001 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3061 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3121 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3181 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3241 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3301 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3361 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3421 cttacaattt aggtggcact tttcggggta cccaggtggc acttttcggg gaaatgtgcg
     3481 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     3541 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     3601 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     3661 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     3721 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     3781 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     3841 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     3901 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     3961 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     4021 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     4081 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     4141 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     4201 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     4261 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     4321 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     4381 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga cctaggtcat
     4441 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4501 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4561 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4621 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4681 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4741 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4801 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4861 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4921 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4981 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5041 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5101 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5161 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5221 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5281 agagcgccat aatccatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     5341 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5401 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     5461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5521 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     5581 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5641 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5701 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     5761 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5821 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5881 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5941 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     6001 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     6061 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     6121 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     6181 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     6241 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     6301 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     6361 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     6421 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     6481 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     6541 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     6601 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     6661 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     6721 acagtccccc ggccacgggg cctgccacca taccca
//
