LOCUS       sequence-217213-        5171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and mCherry (mCherry fluorescent protein,) fusions 
            with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5171)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5171)
  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: 
            "unpublished"
COMMENT     Sequence Label: pExp-mCherry
FEATURES             Location/Qualifiers
     source          1..5171
                     /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    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             131..835
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(273..291)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     541..560
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             888..911
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(925..943)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(926..943)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1001..1019)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1015..1062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1298..1319)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1469..2551)
                     /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     2513..2532
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2552..2629)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2768..2787
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2941..2960)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3060..3082
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3120..3138)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3206..3310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3311..3379,3380..4171)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3529..3548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4342..4930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4831..4850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5084..5101
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggt gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
      181 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
      241 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
      301 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
      361 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
      421 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
      481 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
      541 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
      601 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
      661 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
      721 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
      781 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtctgg
      841 taccgaaaac ctgtacttcc agtgagacct taattaactc gagcgcatgg agccacccgc
      901 agttcgaaaa ataagcttga gtattctata gtgtcaccta aatcccagct tgatccggct
      961 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1021 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1081 tccggataac ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     1141 gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1201 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1261 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1321 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1381 acagcttgca tgcctgcagg tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1441 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1501 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1561 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1621 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1681 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1741 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1801 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1861 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1921 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1981 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2041 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     2101 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2161 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2221 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2281 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2341 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2401 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2461 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2521 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2581 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gcgagctcga
     2641 attgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2701 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2761 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2821 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcctgat
     2881 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     2941 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3001 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3061 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3121 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3181 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3241 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3301 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3361 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3421 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3481 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3541 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3601 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3661 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3721 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3781 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3841 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3901 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3961 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4021 gcgtggttct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4081 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4141 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4201 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4261 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4321 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4381 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4441 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4501 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4561 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4621 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4681 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4741 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4801 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4861 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4921 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4981 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5041 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5101 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5161 atgcagctga t
//
