LOCUS       sequence-126610-        6142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for mCherry purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6142)
  AUTHORS   Niederholtmeyer H, Sun ZZ, Hori Y, Yeung E, Verpoorte A, Murray RM, 
            Maerkl SJ
  TITLE     Rapid cell-free forward engineering of novel genetic ring 
            oscillators.
  JOURNAL   Elife. 2015 Oct 2;4. doi: 10.7554/eLife.09771.
  PUBMED    26430766
REFERENCE   2  (bases 1 to 6142)
  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: "Elife."; 
            date: "2015-10-2"; volume: "4. doi"; pages: " 10.7554/eLife.09771"
COMMENT     Sequence Label: pET21a(+)-Histag-mCherry (527)
FEATURES             Location/Qualifiers
     source          1..6142
                     /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             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             125..835
                     /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(270..288)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     538..557
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             870..887
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(940..958)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      954..1001
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1038..1493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1125..1144)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1335..1356
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1520..1624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1625..1693,1694..2485)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1843..1862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2656..3244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3145..3164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3398..3415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3430..3572
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3498..3517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3658..3680
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3674..3865)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4674..5756)
                     /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     5718..5737
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5757..5834)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6040..6059
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gagaggatcg catcaccatc accatcacgg
      121 atccatggtg agcaagggcg aagaagataa catggccatc atcaaggagt tcatgcgctt
      181 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      241 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      301 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      361 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      421 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      481 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
      541 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
      601 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
      661 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
      721 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
      781 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagagct
      841 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
      901 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      961 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1021 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1081 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1141 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1201 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1261 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1321 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1381 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1441 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcaggtgg
     1501 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1561 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1621 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1681 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1741 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1801 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1861 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1921 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1981 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2041 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2101 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2161 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2221 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2281 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2341 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2401 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2461 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2521 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2581 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2761 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3301 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3361 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3421 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3481 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3541 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3601 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3661 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3721 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3781 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3841 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3901 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3961 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4021 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4081 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4141 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4201 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4261 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4321 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4381 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4441 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4501 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4561 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4621 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4681 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4741 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4801 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4861 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4921 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     4981 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5041 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5101 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5161 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5221 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5281 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5341 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5401 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5461 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5521 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5581 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5641 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5701 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5761 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5821 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5881 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5941 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6001 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6061 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6121 cgagatctcg atcccgcgaa at
//
