LOCUS       sequence-305157-        5318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS3 protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5318)
  AUTHORS   Varasteh Moradi S, Gagoski D, Mureev S, Walden P, McMahon KA, Parton
            RG, Johnston WA, Alexandrov K
  TITLE     Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.
  JOURNAL   J Proteome Res. 2020 Apr 3;19(4):1522-1532. doi: 
            10.1021/acs.jproteome.9b00771. Epub 2020 Mar 9.
  PUBMED    32081002
REFERENCE   2  (bases 1 to 5318)
  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.jproteome.9b00771"; journalName: "J Proteome Res"; 
            date: "2020-04-3- 3"; volume: "19"; issue: "4"; pages: "1522-1532"
COMMENT     Sequence Label: pCellFree_G06_ZIKV NS3
FEATURES             Location/Qualifiers
     source          1..5318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    160..184
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2039..2063)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2065..2088
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2225..2243)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2493..2512
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2797..2820
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      3043..3089
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3167..3183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3167..3183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3180..3202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3191..3207
                     /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)."
     promoter        complement(join(3215..3221,3222..3239,3240..3245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3260..3281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3398..3415)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3569..4157)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3649..3668)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4328..5119,5120..5188))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERTRQIAEIGAS
                     LIKHW"
     primer_bind     4951..4970
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5189..5293)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaat acgactcact atagggacat cttaagttta ttttatttta ttttatttta
       61 ttttatttta ttttatttta ttttatttta ttttatttaa ccatgacagt aatgtataaa
      121 gtctgtaaag acattaaaca cgtaagtgaa accatgggca caagtttgta caaaaaagca
      181 ggcttcagtg gcgccctctg ggacgtgcct gctcccaaag aagtgaagaa aggagagacc
      241 acagatggag tgtacagagt gatgactcgc agactgctag gttcaacaca ggttggagtg
      301 ggagtcatgc aagagggagt cttccacacc atgtggcacg ttacaaaagg agccgcactg
      361 aggagcggtg agggaagact tgatccatac tggggggatg tcaagcagga cttggtgtca
      421 tactgtgggc cttggaagtt ggatgcagct tgggatggac tcagcgaggt acagcttttg
      481 gccgtacctc ccggagagag ggccagaaac attcagaccc tgcctggaat attcaagaca
      541 aaggacgggg acatcggagc agttgctctg gactaccctg cagggacctc aggatctccg
      601 atcctagaca aatgtggaag agtgatagga ctctatggca atggggttgt gatcaagaat
      661 ggaagctatg ttagtgctat aacccaggga aagagggagg aggagactcc ggttgaatgt
      721 ttcgaaccct cgatgctgaa gaagaagcag ctaactgtct tggatctgca tccaggagcc
      781 ggaaaaacca ggagagttct tcctgaaata gtccgtgaag ccataaaaaa gagactccgg
      841 acagtgatct tggcaccaac tagggttgtc gctgctgaga tggaggaggc cttgagagga
      901 cttccggtgc gttacatgac aacagcagtc aacgtcaccc attctgggac agaaatcgtt
      961 gatttgatgt gccatgccac tttcacttca cgcttactac aacccatcag agtccctaat
     1021 tacaatctca acatcatgga tgaagcccac ttcacagacc cctcaagtat agctgcaaga
     1081 ggatacatat caacaagggt tgaaatgggc gaggcggctg ccatttttat gactgccaca
     1141 ccaccaggaa cccgtgatgc gtttcctgac tctaactcac caatcatgga cacagaagtg
     1201 gaagtcccag agagagcctg gagctcaggc tttgattggg tgacagacca ttctgggaaa
     1261 acagtttggt tcgttccaag cgtgagaaac ggaaatgaaa tcgcagcctg tctgacaaag
     1321 gctggaaagc gggtcataca gctcagcagg aagacttttg agacagaatt tcagaaaaca
     1381 aaaaatcaag agtgggactt tgtcataaca actgacatct cagagatggg cgccaacttc
     1441 aaggctgacc gggtcataga ctctaggaga tgcctaaaac cagtcatact tgatggtgag
     1501 agagtcatct tggctgggcc catgcctgtc acgcatgcta gtgctgctca gaggagagga
     1561 cgtataggca ggaaccctaa caaacctgga gatgagtaca tgtatggagg tgggtgtgca
     1621 gagactgatg aaggccatgc acactggctt gaagcaagaa tgcttcttga caacatctac
     1681 ctccaggatg gcctcatagc ctcgctctat cggcctgagg ccgataaggt agccgccatt
     1741 gagggagagt ttaagctgag gacagagcaa aggaagacct tcgtggaact catgaagaga
     1801 ggtgaccttc ccgtctggct agcctatcag gttgcatctg ccggaataac ttacacagac
     1861 agaagatggt gctttgatgg cacaaccaac aacaccataa tggaagacag tgtaccagca
     1921 gaggtttgga caaagtatgg agagaagaga gtgctcaaac cgagatggat ggatgctagg
     1981 gtctgttcag accatgcggc cctgaagtcg ttcaaagaat tcgccgctgg aaaaagaaac
     2041 ccagctttct tgtacaaagt ggtgctggag gtgctcttcc agggcccgaa gggcgaggag
     2101 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     2161 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     2221 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     2281 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     2341 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     2401 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     2461 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gactatgggc
     2521 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     2581 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     2641 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     2701 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     2761 accggcggca tggacgagct gtacaagacc tctggtcacc accatcacca ccatcaccac
     2821 taagcggccg ccctcctcct cctttcttgt tcctttcacg tcgccttctc ggttgtagct
     2881 ggcagacgac gagtcttact tttacgtgta cttctctata gatgatgtat gatctctctg
     2941 catgcgtgtt cgtgcatgtg tccgtgtgtt gggtacgcgt ggtaccctgc aggaaggaag
     3001 ctgagttggc tgctgccacc gctgagcaat aactagtaat tactagcata accccttggg
     3061 gcctctaaac gggtcttgag ggggtttttt gctgaaagga ggacagctga tgattgtcat
     3121 gcttgccatc tgttttcttg caaggtcaga ggaattcgta atcatggtca tagctgtttc
     3181 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3241 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3301 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3361 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3421 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3481 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3541 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3601 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3661 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3721 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3781 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3841 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3901 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3961 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4021 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4081 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4141 gaaaaaaagg atctcaagag gatcctttga tcttttctac ggggtctgac gctcagtgga
     4201 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4261 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4321 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctagttcgtt
     4381 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4441 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4501 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4561 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4621 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4681 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4741 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4801 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4861 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4921 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4981 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5041 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5101 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5161 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5221 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5281 taggggttcc gcgcacattt ccccgaaaag tgccacct
//
