LOCUS       sequence-219912-        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     51..70
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        51..69
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             101..123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             143..172
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             176..208
                     /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(212..220,221..235)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             257..931
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             956..1846
                     /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(1915..1933)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1929..1976
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2073..2528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2160..2179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2370..2391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2554..2658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2659..2727,2728..3519)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPKERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2877..2896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3690..4278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4179..4198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4432..4449
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca ggatctcgat cccgcgaaat taatacgact
       61 cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg
      121 agatatacat atgcggggtt ctcatcatca tcatcatcat catcatcatc atggtatggc
      181 tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataaggatcg
      241 atggggatcc ccgcggatgg cttcctccga agacgttatc aaagagttca tgcgtttcaa
      301 agttcgtatg gaaggttccg ttaacggtca cgagttcgaa atcgaaggtg aaggtgaagg
      361 tcgtccgtac gaaggtaccc agaccgctaa actgaaagtt accaaaggtg gtccgctgcc
      421 gttcgcttgg gacatcctgt ccccgcagtt ccagtacggt tccaaagctt acgttaaaca
      481 cccggctgac atcccggact acctgaaact gtccttcccg gaaggtttca aatgggaacg
      541 tgttatgaac ttcgaagacg gtggtgttgt taccgttacc caggactcct ccctgcaaga
      601 cggtgagttc atctacaaag ttaaactgcg tggtaccaac ttcccgtccg acggtccggt
      661 tatgcagaaa aaaaccatgg gttgggaagc ttccaccgaa cgtatgtacc cggaagacgg
      721 tgctctgaaa ggtgaaatca aaatgcgtct gaaactgaaa gacggtggtc actacgacgc
      781 tgaagttaaa accacctaca tggctaaaaa accggttcag ctgccgggtg cttacaaaac
      841 cgacatcaaa ctggacatca cctcccacaa cgaagactac accatcgttg aacagtacga
      901 acgtgctgaa ggtcgtcact ccaccggtgc ttgtgcggcc gcaggttcgg gttcgatggc
      961 agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
     1021 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
     1081 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     1141 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     1201 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     1261 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     1321 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc
     1381 agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat
     1441 catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac
     1501 tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact
     1561 gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt
     1621 cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac
     1681 cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg
     1741 caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat
     1801 cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt tccggttaat aagaattcga
     1861 agcttgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1921 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1981 aaggaggaac tatatccgga tctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2041 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2101 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2161 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2221 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2281 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2341 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2401 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2461 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2521 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2581 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2641 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2701 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2761 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2821 cttgagagtt ttcgccccaa agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2881 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2941 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3001 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3061 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3121 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3181 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3241 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3301 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3361 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3421 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3481 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3541 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3601 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3661 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3721 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3781 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3841 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3901 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3961 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4021 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4081 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4141 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4201 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4261 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4321 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4381 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4441 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctc
//
