LOCUS       sequence-316023-        6169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of His6_TEV-cleavable-linker_mEos3.2_H-NSdbd in BL21 
            (DE3) pLysS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6169)
  AUTHORS   Rashid FM, Mahlandt E, van der Vaart M, Boer DEC, Varela Alvarez M, 
            Henneman B, Brocken DJW, Voskamp P, Blok AJ, Shimizu TS, Meijer AH, 
            Luijsterburg MS, Goedhart J, Cremazy FGE, Dame RT
  TITLE     HI-NESS: a family of genetically encoded DNA labels based on a 
            bacterial nucleoid-associated protein.
  JOURNAL   Nucleic Acids Res. 2021 Nov 4. pii: 6414584. doi: 
            10.1093/nar/gkab993.
  PUBMED    34734265
REFERENCE   2  (bases 1 to 6169)
  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. 2021 Nov 4. pii: 6414584. doi: 10.1093/nar/gkab993."
COMMENT     Sequence Label: pRD421
FEATURES             Location/Qualifiers
     source          1..6169
                     /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(341..1015)
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="SAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVKE
                     GGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNARN
                     DITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLE
                     GNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPDN
                     ARR"
     CDS             complement(1016..1036)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1043..1060)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1070..1087)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1106..1128
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1143..1167
                     /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(1167..1186)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1168..1186)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1270..1289)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1495..1572
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1573..2655
                     /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(1592..1611)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3464..3655
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3649..3671)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3757..3899
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3812..3831
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3914..3931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4085..4673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4165..4184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4784..4803)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4795..5610
                     /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             4795..5610
                     /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(4868..4887)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5703..6158)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5840..5861)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6052..6071
                     /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 gagttattgc ttgatcagga
      181 aatcgtcgag ggatttacct tgctcatcca ttgctttttt gattacagct ggagtacggc
      241 cttggccagt ccaggtttta gtttcgccgt tttcgtcaac gtagctatat tttgccggac
      301 gctgagcacg tttagctttg gtgccagatt taacggcagc tcgtcttgca ttgtcaggca
      361 atccagaatg agcaacagca tgctcataca gcttaacctt gttgtaatct ttgtcatggc
      421 ttaaaatctc aatgcagtgg tccacaaagt gggcgcctgg taacttgaca cccttctcct
      481 tagctttgta agtagttctg aagtcacatc ggtaatgggc atttccttca agcaacaaag
      541 ccatctcaat atcacccgtc agcactccat cacgcacata cattttctca gtggagggct
      601 cccatttcag cgtcttcttc tgcataactg gaccattggc gggaaagttg gtaccataaa
      661 atcgaacttt attatagaaa gtgtcccctt ccattgttat gtcgtttctg gcgttgcaaa
      721 tgcccccgtc ttcgaaagtc aagcttcgtt cccacgaata ccccttagga aacgactgct
      781 taaaatagtc ttgtatgttg tctggatatt tggcgaatac cctgttgccg taatggaatg
      841 cagtggtcag gatatcaaag gcaaaaggca gaggtccgcc ctctttgact tcaagatcca
      901 tactctgttt tccctcaaaa ggcttgcctg taccatctcc gtcgatcaca aagtggtgcc
      961 cgtttacgtt gccttccata cggagtttga tcttcatgtc tggcttaatc gcactaccct
     1021 gaaaatacag gttttccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat
     1081 gatgatggct gctgcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     1141 ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg
     1201 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     1261 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     1321 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1381 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     1441 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     1501 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     1561 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     1621 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     1681 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1741 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1801 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     1861 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1921 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     1981 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2041 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2101 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2161 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2221 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2281 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2341 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     2401 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     2461 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     2521 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     2581 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2641 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     2701 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     2761 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     2821 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     2881 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     2941 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     3001 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3061 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3121 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3181 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3241 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3301 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3361 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     3421 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     3481 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     3541 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     3601 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3661 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3721 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3781 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3841 caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     3901 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3961 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4021 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4081 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4141 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4201 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4261 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4321 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4381 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4441 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4501 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4561 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4621 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4681 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4741 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4801 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     4861 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     4921 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     4981 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     5041 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5101 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5161 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5221 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5281 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5341 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     5401 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     5461 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     5521 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     5581 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     5641 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5701 tgaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5761 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5821 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5881 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5941 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     6001 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     6061 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     6121 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
