LOCUS       sequence-226686-        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     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1931..2641
                     /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(2076..2094)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2344..2363
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2652..2669
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2722..2740)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2736..2783
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2820..3275
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2907..2926)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3117..3138
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3390..4205)
                     /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(3390..4205)
                     /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     4113..4132
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4197..4216
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4358..4946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4847..4866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5100..5117
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5288..6370)
                     /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     6332..6351
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6371..6448)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6654..6673
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggatcccac catcatcacc accatgccat
      121 ggccacggac gcaattgacc actactatca gaacagggat ggaccaccgt cgagatcctt
      181 cagcgatgag tacgattcat acaacaaaac cgcccctttg cctccactat cagcatgtcc
      241 tgaaagagcc cagactccaa attcaaagcg tattccgata acccccaatt ctgccaaatc
      301 tacatctgag agcccacgcc gcatcacttt ttctagggat gccggaagta gcacaaaagg
      361 ctcgacacaa acaagtccta gtaaccaagg aagtgtaagc agtaggaatg gtagcaatgg
      421 aaatggttta gggctgagta gcgcgggaaa ctttgcagat ttctttagct ctgaagtgtt
      481 ttatattgtg cttcataatc caacgaccgc gcatcgattc ttgaagtttt gtcaaagtcg
      541 tgcttgttcg gaaaaccttg aatttttgca aaagattgat gagtataatc gattaataga
      601 cgaagcgaca cgtcttcttt ccacgattta ctcaacctat gtttcgtcag aagcaccaaa
      661 gcaaatcaat atttccaatt ctcatgctcg acgattatca catgatatca agggtgcgac
      721 gcataatgtt cttcccggcc ttgaagacat cttcaatggg tcccaggagc aaattgagaa
      781 attgctagct agtgatttgt atccgcgttt tgtaaaacac caggttactg cctccgcgac
      841 tatggcgttg gcaaatgata aggaaagata tccaggtcta ggtgactgtt tttgcttgac
      901 tgatccaagg cgagccgata acccaattgt atttgcttcg gacgggttcg tctctgtcac
      961 gggatattcc cgatcagata taatacctcg aaactgtcgt ttcctacaag gatcgtttac
     1021 cgatcgacaa gcaaccaaac gtctacgaac ttccattgaa aaccgtgaag agacagttga
     1081 attacttctg aattatcgca aaaatggcga tccattctgg aatctccttt acgtatctcc
     1141 tttacttgac ggtaatgggg acgtcagatt ctttctcggt ggaaacatca attgcagtac
     1201 gactattcac agtcgcaccg atgttcttcg aattttgagt ctgaacgatg aggacactgg
     1261 gagctttata gaggggagta acaagacccc gagtgttcga agcaaggagt ccaataatgc
     1321 aagcaactat gggaaaacct cattcttcaa gagcttcaaa aagtacaagg cgaatactgt
     1381 agaaatccga gacgaagctg ggatggaagg cgaattgatc gatcggatcg gaaagctaca
     1441 attcaaaaca caagttgaag agttctatac ggcttattca aagtatcttg tcctatctta
     1501 ccaaccacag tcacaaaatc tgattgtcaa atacttctct ccgggtatag tggacatgct
     1561 taatctaaac atgcccaatg gcctcgtagc tcctattgtc gataaagata tcttcaaagt
     1621 tctcacagaa cactccccgt cgtctgtacc gcgcaccttc aaaaatgtgg ttagagaagg
     1681 tatcaaagcc ggacgagcag tatcagtcga aacgggactt ctgactggaa ttgaagagat
     1741 caagaagtca caaaatattt ttagtggcac aacggcaaat agagagagag ggtggaagaa
     1801 ggccgaggaa aaatatgtct gtcattttac accttgtaaa gatgagatgg gcagagttgg
     1861 atgggtcgtt ttgactgtgg cgccgaaact ggagagattt gaattcggtg gtggatcagg
     1921 tggaggatct atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     1981 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     2041 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     2101 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     2161 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     2221 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     2281 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     2341 cggccccgta atgcagaaga agactatggg ctgggaggcc tcctccgagc ggatgtaccc
     2401 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     2461 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     2521 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     2581 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     2641 atgaactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     2701 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2761 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2821 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2881 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2941 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3001 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3061 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3121 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3181 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3241 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggtacccag
     3301 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3361 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     3421 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3481 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3541 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3601 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3661 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3721 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3781 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3841 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3901 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3961 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     4021 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     4081 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     4141 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     4201 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     4261 aatcccttaa cgtgacctag gtcatgacca aaatccctta acgtgagttt tcgttccact
     4321 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4381 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4441 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4501 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4561 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4621 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4681 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4741 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4801 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4861 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4921 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4981 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5041 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5101 gcgagtcagt gagcgaggaa gcggaagagc gccataatcc atgataaaga agacagtcat
     5161 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5221 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5281 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5341 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5401 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5461 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5521 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5581 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5641 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5701 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5761 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5821 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5881 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5941 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6001 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6061 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6121 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6181 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6241 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6301 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6361 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6421 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6481 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6541 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6601 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6661 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6721 ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
