LOCUS       sequence-308740-        3848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS4A protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3848)
  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 3848)
  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 NS4A
FEATURES             Location/Qualifiers
     source          1..3848
                     /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(569..593)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             595..618
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(755..773)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1023..1042
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1327..1350
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      1573..1619
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1697..1713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1697..1713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1710..1732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1721..1737
                     /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(1745..1751,1752..1769,1770..1775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1790..1811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1928..1945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2099..2687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2179..2198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2858..3649,3650..3718))
                     /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     3481..3500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3719..3823)
                     /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 ggcttcggag cggctttggg agtaatggag gccctgggaa cactgccagg acacatgaca
      241 gagaggtttc aggaagccat tgacaacctc gccgtgctca tgcgagcaga gactggaagc
      301 aggccttata aggcagcggc agcccaactg ccggagaccc tagagacgat tatgctctta
      361 ggtttgctgg gaacagtttc actggggatc ttcttcgtct tgatgcggaa taagggcatc
      421 gggaagatgg gctttggaat ggtaaccctt ggggccagtg catggctcat gtggctttcg
      481 gaaattgaac cagccagaat tgcatgtgtc ctcattgttg tgtttttatt actggtggtg
      541 ctcatacccg agccagagaa gcaaagaaac ccagctttct tgtacaaagt ggtgctggag
      601 gtgctcttcc agggcccgaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      661 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      721 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
      781 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
      841 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
      901 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
      961 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1021 ggccccgtaa tgcagaagaa gactatgggc tgggaggcct cctccgagcg gatgtacccc
     1081 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1141 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1201 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1261 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagacc
     1321 tctggtcacc accatcacca ccatcaccac taagcggccg ccctcctcct cctttcttgt
     1381 tcctttcacg tcgccttctc ggttgtagct ggcagacgac gagtcttact tttacgtgta
     1441 cttctctata gatgatgtat gatctctctg catgcgtgtt cgtgcatgtg tccgtgtgtt
     1501 gggtacgcgt ggtaccctgc aggaaggaag ctgagttggc tgctgccacc gctgagcaat
     1561 aactagtaat tactagcata accccttggg gcctctaaac gggtcttgag ggggtttttt
     1621 gctgaaagga ggacagctga tgattgtcat gcttgccatc tgttttcttg caaggtcaga
     1681 ggaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1741 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1801 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1861 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1921 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1981 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2041 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2101 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2161 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2221 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2281 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2341 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2401 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2461 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2521 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2581 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2641 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagag gatcctttga
     2701 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2761 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2821 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2881 cacctatctc agcgatctgt ctagttcgtt catccatagt tgcctgactc cccgtcgtgt
     2941 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3001 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3061 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3121 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3181 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3241 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3301 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3361 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3421 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3481 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3541 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3601 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3661 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3721 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3781 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3841 tgccacct
//
