LOCUS       sequence-217232-        6453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 2(KW)2-mCherry in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6453)
  AUTHORS   Lee S, Oh Y, Lee J, Choe S, Lim S, Lee HS, Jo K, Schwartz DC
  TITLE     DNA binding fluorescent proteins for the direct visualization of 
            large DNA molecules.
  JOURNAL   Nucleic Acids Res. 2016 Jan 8;44(1):e6. doi: 10.1093/nar/gkv834. 
            Epub 2015 Aug 11.
  PUBMED    26264666
REFERENCE   2  (bases 1 to 6453)
  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: "Nucleic 
            Acids Res."; date: "2016-01-8"; volume: "44(1):e6. doi"; pages: " 
            10.1093/nar/gkv834. Epub 2015 Aug 11"
COMMENT     Sequence Label: pET-2(KW)2-mCherry
FEATURES             Location/Qualifiers
     source          1..6453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..34)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        102..206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(207..275,276..1067)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(425..444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1238..1826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1727..1746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1980..1997
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2012..2151
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2077..2096)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2237..2259
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2253..2444)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3756..4838)
                     /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     4800..4819
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4839..4916)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5122..5141
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5225..5244
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5225..5243
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5244..5268
                     /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             5283..5305
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5324..5341
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5351..5368
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5396..6103
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDQMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5541..5559)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5809..5828
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6003..6026
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(6178..6196)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6192..6239
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6414..6419,6420..6436,6437..6442))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
       61 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      121 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      181 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      241 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      301 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      361 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      421 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
      481 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
      541 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
      601 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
      661 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
      721 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
      781 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
      841 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
      901 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
      961 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1021 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1081 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1141 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1201 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1261 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1321 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1381 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1441 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1501 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1561 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1621 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1681 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1741 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1801 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1861 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1921 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     1981 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2041 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2101 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     2161 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2221 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2281 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     2341 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     2401 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     2461 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     2521 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     2581 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     2641 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     2701 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     2761 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     2821 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     2881 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     2941 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     3001 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     3061 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     3121 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     3181 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     3241 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     3301 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     3361 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     3421 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3481 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3541 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3601 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3661 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3721 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3901 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3961 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4021 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4081 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4141 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4201 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4261 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4321 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4381 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4441 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4501 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4561 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4621 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4681 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4741 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4801 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4861 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4921 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4981 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5041 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5101 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5161 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     5221 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5281 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     5341 cagcagcggc ctggtgccgc gcggcagcca tatgaaatgg aaatggaaaa aagcgatggt
     5401 gagcaagggc gaggaggacc agatggccat catcaaggag ttcatgcgct tcaaggtgca
     5461 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     5521 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc
     5581 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     5641 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     5701 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     5761 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     5821 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     5881 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgccgaggt
     5941 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtgaacat
     6001 caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgcgc
     6061 cgagggccgc cactccaccg gcggcatgga cgagctgtac aaggcgaaaa aatggaaatg
     6121 gaaataagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     6181 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     6241 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa
     6301 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
     6361 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
     6421 cttatcgatg ataagctgtc aaacatgaga att
//
