LOCUS       sequence-210559-        4483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His10-RFP-Halo; construct generated for pull-down and 
            localization experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4483)
  AUTHORS   Caldwell RM, Bermudez JG, Thai D, Aonbangkhen C, Schuster BS, 
            Courtney T, Deiters A, Hammer DA, Chenoweth DM, Good MC
  TITLE     Optochemical Control of Protein Localization and Activity within 
            Cell-like Compartments.
  JOURNAL   Biochemistry. 2018 May 8;57(18):2590-2596. doi: 
            10.1021/acs.biochem.8b00131. Epub 2018 Apr 19.
  PUBMED    29671583
REFERENCE   2  (bases 1 to 4483)
  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"; doi: 
            "10.1021/acs.biochem.8b00131"; journalName: "Biochemistry"; date: 
            "2018-05-8- 8"; volume: "57"; issue: "18"; pages: "2590-2596"
COMMENT     Sequence Label: pJB01
FEATURES             Location/Qualifiers
     source          1..4483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..141
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             145..177
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(181..189,190..204)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             226..900
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             925..1815
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(1884..1902)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1898..1945
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2042..2497
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2129..2148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2339..2360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2523..2627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2628..2696,2697..3488)
                     /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(2846..2865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3659..4247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4148..4167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4401..4418
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatc atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg
      181 gatctgtacg acgatgacga taaggatcga tggggatccc cgcggatggc ttcctccgaa
      241 gacgttatca aagagttcat gcgtttcaaa gttcgtatgg aaggttccgt taacggtcac
      301 gagttcgaaa tcgaaggtga aggtgaaggt cgtccgtacg aaggtaccca gaccgctaaa
      361 ctgaaagtta ccaaaggtgg tccgctgccg ttcgcttggg acatcctgtc cccgcagttc
      421 cagtacggtt ccaaagctta cgttaaacac ccggctgaca tcccggacta cctgaaactg
      481 tccttcccgg aaggtttcaa atgggaacgt gttatgaact tcgaagacgg tggtgttgtt
      541 accgttaccc aggactcctc cctgcaagac ggtgagttca tctacaaagt taaactgcgt
      601 ggtaccaact tcccgtccga cggtccggtt atgcagaaaa aaaccatggg ttgggaagct
      661 tccaccgaac gtatgtaccc ggaagacggt gctctgaaag gtgaaatcaa aatgcgtctg
      721 aaactgaaag acggtggtca ctacgacgct gaagttaaaa ccacctacat ggctaaaaaa
      781 ccggttcagc tgccgggtgc ttacaaaacc gacatcaaac tggacatcac ctcccacaac
      841 gaagactaca ccatcgttga acagtacgaa cgtgctgaag gtcgtcactc caccggtgct
      901 tgtgcggccg caggttcggg ttcgatggca gaaatcggta ctggctttcc attcgacccc
      961 cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc
     1021 acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg caacatcatc
     1081 ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat gggcaaatcc
     1141 gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga tgccttcatc
     1201 gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc cgctctgggt
     1261 ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat ggagttcatc
     1321 cgccctatcc cgacctggga cgaatggcca gaatttgccc gcgagacctt ccaggccttc
     1381 cgcaccaccg acgtcggccg caagctgatc atcgatcaga acgtttttat cgagggtacg
     1441 ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta ccgcgagccg
     1501 ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct gccaatcgcc
     1561 ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct gcaccagtcc
     1621 cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc
     1681 gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc gggtctgaat
     1741 ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg gctgtcgacg
     1801 ctcgagattt ccggttaata agaattcgaa gcttgatccg gctgctaaca aagcccgaaa
     1861 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1921 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat
     1981 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2041 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2101 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2161 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2221 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2281 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2341 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2401 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2461 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2521 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2581 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2641 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2701 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2761 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2821 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2881 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2941 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3001 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3061 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     3841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     4441 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cag
//
