LOCUS       sequence-305820-        4523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS1 protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4523)
  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 4523)
  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 NS1
FEATURES             Location/Qualifiers
     source          1..4523
                     /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(1244..1268)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1270..1293
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1430..1448)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1698..1717
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2002..2025
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2248..2294
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2372..2388)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2372..2388)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2385..2407)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2396..2412
                     /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(2420..2426,2427..2444,2445..2450))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2465..2486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2603..2620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2774..3362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2854..2873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3533..4324,4325..4393))
                     /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     4156..4175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4394..4498)
                     /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 ggcttcgacg tggggtgctc agtggacttc tcaaaaaagg aaacgagatg tggcacgggg
      241 gtattcatct ataatgatgt tgaagcctgg agggaccggt acaagtacca tcctgactcc
      301 ccccgcagat tggcagcagc agtcaagcag gcctgggaag aggggatctg tgggatctca
      361 tccgtttcaa gaatggaaaa catcatgtgg aaatcagtag aaggggagct caatgctatc
      421 ctagaggaga atggagttca actgacagtt gttgtgggat ctgtaaaaaa ccccatgtgg
      481 agaggtccac aaagattgcc agtgcctgtg aatgagctgc cccacggctg gaaagcctgg
      541 gggaaatcgt attttgttag ggcggcaaag accaacaaca gttttgttgt cgacggtgac
      601 acactgaagg aatgtccgct tgagcacaga gcatggaata gttttcttgt ggaggatcac
      661 gggtttggag tcttccacac cagtgtctgg cttaaggtca gagaagatta ctcattagaa
      721 tgtgacccag ccgtcatagg aacagctgtt aagggaaggg aggccgcgca cagtgatctg
      781 ggctattgga ttgaaagtga aaagaatgac acatggaggc tgaagagggc ccacctgatt
      841 gagatgaaaa catgtgaatg gccaaagtct cacacattgt ggacagatgg agtagaagaa
      901 agtgatctta tcatacccaa gtctttagct ggtccactca gccaccacaa caccagagag
      961 ggttacagaa cccaagtgaa agggccttgg cacagtgaag agcttgaaat ccggtttgag
     1021 gaatgtccag gcaccaaggt ttacgtggag gagacatgcg gaactagagg accatctctg
     1081 agatcaacta ctgcaagtgg aagggtcatt gaggaatggt gctgtaggga atgcacaatg
     1141 cccccactat cgtttcgagc aaaagacggc tgctggtatg gaatggagat aaggcccagg
     1201 aaagaaccag agagcaactt agtgaggtca atggtgacag cgaacccagc tttcttgtac
     1261 aaagtggtgc tggaggtgct cttccagggc ccgaagggcg aggaggataa catggccatc
     1321 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     1381 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     1441 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     1501 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     1561 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     1621 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     1681 aacttcccct ccgacggccc cgtaatgcag aagaagacta tgggctggga ggcctcctcc
     1741 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     1801 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     1861 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     1921 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     1981 gagctgtaca agacctctgg tcaccaccat caccaccatc accactaagc ggccgccctc
     2041 ctcctccttt cttgttcctt tcacgtcgcc ttctcggttg tagctggcag acgacgagtc
     2101 ttacttttac gtgtacttct ctatagatga tgtatgatct ctctgcatgc gtgttcgtgc
     2161 atgtgtccgt gtgttgggta cgcgtggtac cctgcaggaa ggaagctgag ttggctgctg
     2221 ccaccgctga gcaataacta gtaattacta gcataacccc ttggggcctc taaacgggtc
     2281 ttgagggggt tttttgctga aaggaggaca gctgatgatt gtcatgcttg ccatctgttt
     2341 tcttgcaagg tcagaggaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2401 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2461 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2521 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2581 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2641 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2701 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2761 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2821 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2881 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2941 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3001 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3061 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3361 aagaggatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3541 gcttaatcag tgaggcacct atctcagcga tctgtctagt tcgttcatcc atagttgcct
     3601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4501 catttccccg aaaagtgcca cct
//
