LOCUS       sequence-305155-        4220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS4B protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4220)
  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 4220)
  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 NS4B
FEATURES             Location/Qualifiers
     source          1..4220
                     /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(941..965)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             967..990
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1127..1145)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1395..1414
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1699..1722
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      1945..1991
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2069..2085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2069..2085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2082..2104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2093..2109
                     /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(2117..2123,2124..2141,2142..2147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2162..2183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2300..2317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2471..3059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2551..2570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3230..4021,4022..4090))
                     /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     3853..3872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4091..4195)
                     /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 ggcttcaacg aacttggatg gctggaaaga acaaaaaatg acatagctca tctaatggga
      241 aggagagaag aaggagcaac gatgggattc tcaatggaca ttgatctgcg gccagcctcc
      301 gcctgggcta tctatgccgc attgacaact ctcatcaccc cagctgtcca acatgcggta
      361 accacttcat acaacaacta ctccttaatg gcgatggcca cacaagctgg agtgctgttt
      421 ggcatgggca aagggatgcc atttatgcat ggggaccttg gagtcccgct gctaatgatg
      481 ggttgctatt cacaattaac acccctgact ctgatagtag ctatcattct gcttgtggcg
      541 cactacatgt acttgatccc aggcctacaa gcggcagcag cgcgtgctgc ccagaaaagg
      601 acagcagctg gcatcatgaa gaatcccgtt gtggatggaa tagtggtaac tgacattgac
      661 acaatgacaa tagaccccca ggtggagaag aagatgggac aagtgttact catagcagta
      721 gccatctcca gtgctgtgct gctgcggacc gcctggggat ggggggaggc tggagctctg
      781 atcacagcag cgacctccac cttgtgggaa ggctctccaa acaaatactg gaactcctct
      841 acagccacct cactgtgcaa catcttcaga ggaagctatc tggcaggagc ttcccttatc
      901 tatacagtga cgagaaacgc tggcctggtt aagagacgta acccagcttt cttgtacaaa
      961 gtggtgctgg aggtgctctt ccagggcccg aagggcgagg aggataacat ggccatcatc
     1021 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1081 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1141 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     1201 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     1261 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     1321 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     1381 ttcccctccg acggccccgt aatgcagaag aagactatgg gctgggaggc ctcctccgag
     1441 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     1501 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     1561 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     1621 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     1681 ctgtacaaga cctctggtca ccaccatcac caccatcacc actaagcggc cgccctcctc
     1741 ctcctttctt gttcctttca cgtcgccttc tcggttgtag ctggcagacg acgagtctta
     1801 cttttacgtg tacttctcta tagatgatgt atgatctctc tgcatgcgtg ttcgtgcatg
     1861 tgtccgtgtg ttgggtacgc gtggtaccct gcaggaagga agctgagttg gctgctgcca
     1921 ccgctgagca ataactagta attactagca taaccccttg gggcctctaa acgggtcttg
     1981 agggggtttt ttgctgaaag gaggacagct gatgattgtc atgcttgcca tctgttttct
     2041 tgcaaggtca gaggaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2101 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2161 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2221 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2821 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3061 aggatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3241 taatcagtga ggcacctatc tcagcgatct gtctagttcg ttcatccata gttgcctgac
     3301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3361 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3421 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3481 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3541 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3601 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3661 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3721 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3781 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3841 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3901 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3961 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4021 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4081 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4141 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4201 ttccccgaaa agtgccacct
//
