LOCUS       sequence-257347-        6130 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression vector for mCherry fused to a tandem repeat of the E. 
            coli MinD membrane targeting sequence (MTS) (C-terminal fusion) and 
            an N-terminal His-tag for purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6130)
  AUTHORS   Ramm B, Glock P, Mucksch J, Blumhardt P, Garcia-Soriano DA, Heymann 
            M, Schwille P
  TITLE     The MinDE system is a generic spatial cue for membrane protein 
            distribution in vitro.
  JOURNAL   Nat Commun. 2018 Sep 26;9(1):3942. doi: 10.1038/s41467-018-06310-1.
  PUBMED    30258191
REFERENCE   2  (bases 1 to 6130)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-09-26"; volume: "9(1):3942. doi"; pages: " 
            10.1038/s41467-018-06310-1"
COMMENT     Sequence Label: pET28a-mCherry-2xEcMTS
FEATURES             Location/Qualifiers
     source          1..6130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(290..997)
                     /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(565..584)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     834..852
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1031..1048)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1067..1089
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1104..1128
                     /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)."
     primer_bind     complement(1128..1147)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1129..1147)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1231..1250)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1456..1533
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1534..2616
                     /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     complement(1553..1572)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3425..3616
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3610..3632)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3718..3860
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3773..3792
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3875..3892)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4046..4634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4126..4145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4745..4764)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4756..5571
                     /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             4756..5571
                     /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     complement(4829..4848)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5664..6119)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5801..5822)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6013..6032
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
      181 atcctccgaa caagcgtttg aggaagcctt tcttctcttc ttcaatagat ccgcctcctc
      241 cgaacaagcg tttgaggaag cctttcttct cttcttcaat gccagatccc ttgtacagct
      301 cgtccatgcc gccggtggag tggcggccct cggcgcgttc gtactgttcc acgatggtgt
      361 agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg ccgggcagct
      421 gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct
      481 tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg tacatccgct
      541 cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg gaggggaagt
      601 tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag gagtcctggg
      661 tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg
      721 ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag gccttggagc
      781 cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca cccttggtca
      841 ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct
      901 cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg aactccttga
      961 tgatggccat gttatcctcc tcgcccttgc tcaccatcct aggacttcca cctgaacctc
     1021 cagatccacc gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag
     1081 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     1141 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     1201 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     1261 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     1321 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     1381 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     1441 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     1501 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     1561 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     1621 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     1681 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     1741 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     1801 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     1861 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     1921 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     1981 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     2041 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     2101 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     2161 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     2221 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     2281 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     2341 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     2401 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     2461 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     2521 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2581 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     2641 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     2701 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     2761 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     2821 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     2881 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     2941 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     3001 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     3061 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     3121 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     3181 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     3241 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3301 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3361 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     3421 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3481 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3541 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3601 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3661 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3721 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3781 gagcagattg tactgagagt gcaccatata tgcggtgtga aataccgcac agatgcgtaa
     3841 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3901 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3961 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4021 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4081 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4141 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4201 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4261 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4321 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4381 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4441 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4501 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4561 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4621 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4681 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     4741 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     4801 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     4861 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     4921 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     4981 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     5041 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     5101 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     5161 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     5221 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     5281 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     5341 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5401 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5461 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5521 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     5581 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5641 atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa aattcgcgtt
     5701 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5761 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5821 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5881 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5941 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6001 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6061 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6121 ccattcgcca
//
