LOCUS       sequence-313307-        4145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS2A protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4145)
  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 4145)
  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 NS2A
FEATURES             Location/Qualifiers
     source          1..4145
                     /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(866..890)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             892..915
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1052..1070)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1320..1339
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1624..1647
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      1870..1916
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1994..2010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1994..2010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2007..2029)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2018..2034
                     /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(2042..2048,2049..2066,2067..2072))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2087..2108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2225..2242)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2396..2984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2476..2495)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3155..3946,3947..4015))
                     /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     3778..3797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4016..4120)
                     /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 ggcttcgggt caaccgatca tatggaccac ttctctcttg gagtgcttgt gattctactc
      241 atggtgcagg aggggttgaa gaagagaatg accacaaaga tcatcatgag cacatcaatg
      301 gcagtgctgg tagtcatgat cttgggagga ttttcaatga gtgacctggc caagcttgtg
      361 atcctgatgg gtgctacttt cgcagaaatg aacactggag gagatgtagc tcacttggca
      421 ttggtagcgg catttaaagt cagaccagcc ttgctggtct ccttcatttt cagagccaat
      481 tggacacccc gtgagagcat gctgctagcc ctggcttcgt gtcttctgca aactgcgatc
      541 tctgctcttg aaggtgactt gatggtcctc attaatggat ttgctttggc ctggttggca
      601 attcgagcaa tggccgtgcc acgcactgac aacatcgctc taccaatctt ggctgctcta
      661 acaccactag ctcgaggcac actgctcgtg gcatggagag cgggcctggc tacttgtgga
      721 gggatcatgc tcctctccct gaaagggaaa ggtagtgtga agaagaacct gccatttgtc
      781 atggccctgg gattgacagc tgtgagggta gtagacccta ttaatgtggt aggactactg
      841 ttactcacaa ggagtgggaa gcggaaccca gctttcttgt acaaagtggt gctggaggtg
      901 ctcttccagg gcccgaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
      961 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1021 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1081 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1141 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1201 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1261 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1321 cccgtaatgc agaagaagac tatgggctgg gaggcctcct ccgagcggat gtaccccgag
     1381 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1441 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1501 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1561 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagacctct
     1621 ggtcaccacc atcaccacca tcaccactaa gcggccgccc tcctcctcct ttcttgttcc
     1681 tttcacgtcg ccttctcggt tgtagctggc agacgacgag tcttactttt acgtgtactt
     1741 ctctatagat gatgtatgat ctctctgcat gcgtgttcgt gcatgtgtcc gtgtgttggg
     1801 tacgcgtggt accctgcagg aaggaagctg agttggctgc tgccaccgct gagcaataac
     1861 tagtaattac tagcataacc ccttggggcc tctaaacggg tcttgagggg gttttttgct
     1921 gaaaggagga cagctgatga ttgtcatgct tgccatctgt tttcttgcaa ggtcagagga
     1981 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2041 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2101 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2161 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2221 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2281 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2341 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2401 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2461 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2521 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2581 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2641 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2701 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2761 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2821 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2881 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2941 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaggat cctttgatct
     3001 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3061 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3121 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3181 ctatctcagc gatctgtcta gttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3241 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3301 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3361 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3421 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3481 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3541 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3601 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3661 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3721 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3781 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3841 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3901 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3961 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4021 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4081 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4141 cacct
//
