LOCUS       sequence-305162-        8673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS5 protein 
            with N-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8673)
  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 8673)
  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: pmCellFree _KA2_ZIKV NS5
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2147..2166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2147..2165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2309..2332
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2339..3040
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(2478..2496)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2746..2765
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3041..3064
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3065..3089
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5802..5826)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5861..5878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5968..5987)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6033..6088
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6087..6106)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      6176..6223
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     6236..6941
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           6324..6696
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(6353..6941)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6950..6983)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(7151..7738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7230..7249)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7751..8542,8543..8611))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8374..8393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccgg gggggggggg gggggggggg gggggggggg ggggggggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gacgtctaat acgactcact
     2161 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
     2221 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
     2281 cgtaagtgaa accatgggca cctctggtca ccaccatcac caccatcacc acggcacaag
     2341 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     2401 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2461 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     2521 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     2581 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     2641 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     2701 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     2761 gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     2821 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     2881 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     2941 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     3001 gggccgccac tccaccggcg gcatggacga gctgtacaag ctggaggtgc tcttccaggg
     3061 tccgacaagt ttgtacaaaa aagcaggctt cggaggtggg acgggagaga ctctgggaga
     3121 gaagtggaaa gctcgtctga atcagatgtc ggccctggag ttctactctt ataaaaagtc
     3181 aggtatcact gaagtgtgta gagaggaggc tcgccgtgcc ctcaaggatg gagtggccac
     3241 aggaggacat gccgtatccc ggggaagtgc aaagatcaga tggttggagg agagaggata
     3301 tctgcagccc tatgggaagg ttgttgacct cggatgtggc agagggggct ggagctatta
     3361 tgccgccacc atccgcaaag tgcaggaggt gagaggatac acaaagggag gtcccggtca
     3421 tgaagaaccc atgctggtgc aaagctatgg gtggaacata gttcgtctca agagtggagt
     3481 ggacgtcttc cacatggcgg ctgagccgtg tgacactctg ctgtgtgaca taggtgagtc
     3541 atcatctagt cctgaagtgg aagagacacg aacactcaga gtgctctcta tggtggggga
     3601 ctggcttgaa aaaagaccag gggccttctg tataaaggtg ctgtgcccat acaccagcac
     3661 tatgatggaa acgatggagc gactgcaacg taggcatggg ggaggattag tcagagtgcc
     3721 attgtgtcgc aactccacac atgagatgta ctgggtctct ggggcaaaga gcaacatcat
     3781 aaaaagtgtg tccaccacaa gtcagctcct cctgggacgc atggatggcc ccaggaggcc
     3841 agtgaaatat gaggaggatg tgaacctcgg ctcgggtaca cgagctgtgg caagctgtgc
     3901 tgaggctcct aacatgaaaa tcatcggcag gcgcattgag agaatccgca atgaacatgc
     3961 agaaacatgg tttcttgatg aaaaccaccc atacaggaca tgggcctacc acgggagcta
     4021 cgaagccccc acgcaaggat cagcgtcttc cctcgtgaac ggggttgtta gactcctgtc
     4081 aaagccttgg gacgtggtga ctggagttac aggaatagcc atgactgaca ccacaccata
     4141 cggccaacaa agagtcttca aagaaaaagt ggacaccagg gtgccagatc cccaagaagg
     4201 cactcgccag gtaatgaaca tagtctcttc ctggctgtgg aaggagctgg ggaaacgcaa
     4261 gcggccacgc gtctgcacca aagaagagtt tatcaacaag gtgcgcagca atgcagcact
     4321 gggagcaata tttgaagagg aaaaagaatg gaagacggct gtggaagctg tgaatgatcc
     4381 aaggttttgg gccctagtgg atagggagag agaacaccac ctgagaggag agtgtcacag
     4441 ctgtgtgtac aacatgatgg gaaaaagaga aaagaagcaa ggagagttcg ggaaagcaaa
     4501 aggtagccgc gccatctggt acatgtggtt gggagccaga ttcttggagt ttgaagccct
     4561 tggattcttg aacgaggacc attggatggg aagagaaaac tcaggaggtg gagtcgaagg
     4621 gttaggattg caaagacttg gatacattct agaagaaatg aatcgggcac caggaggaaa
     4681 gatgtacgca gatgacactg ctggctggga cacccgcatt agtaagtttg atctggagaa
     4741 tgaagctctg attaccaacc aaatggagga agggcacaga actctggcgt tggccgtgat
     4801 taaatacaca taccaaaaca aagtggtgaa ggttctcaga ccagctgaag gaggaaaaac
     4861 agttatggac atcatttcaa gacaagacca gagagggagt ggacaagttg tcacttatgc
     4921 tctcaacaca ttcaccaact tggtggtgca gcttatccgg aacatggaag ctgaggaagt
     4981 gttagagatg caagacttat ggttgttgag gaagccagag aaagtgacca gatggttgca
     5041 gagcaatgga tgggatagac tcaaacgaat ggcggtcagt ggagatgact gcgttgtgaa
     5101 gccaatcgat gataggtttg cacatgccct caggttcttg aatgacatgg gaaaagttag
     5161 gaaagacaca caggagtgga aaccctcgac tggatggagc aattgggaag aagtcccgtt
     5221 ctgctcccac cacttcaaca agctgtacct caaggatggg agatccattg tggtcccttg
     5281 ccgccaccaa gatgaactga ttggccgagc tcgcgtctca ccaggggcag gatggagcat
     5341 ccgggagact gcctgtcttg caaaatcata tgcgcagatg tggcagctcc tttatttcca
     5401 cagaagagat cttcgactga tggctaatgc catttgctcg gctgtgccag ttgactgggt
     5461 accaactggg agaaccacct ggtcaatcca cggaaaggga gaatggatga ccactgagga
     5521 catgctcatg gtgtggaata gagtgtggat tgaggagaac gaccatatgg aggacaagac
     5581 tcctgtaaca aaatggacag acattcccta tctaggaaaa agggaggact tatggtgtgg
     5641 atcccttata gggcacagac cccgcaccac ttgggctgaa aacatcaaag acacagtcaa
     5701 catggtgcgc aggatcatag gtgatgaaga aaagtacatg gactatctat ccacccaagt
     5761 ccgctacttg ggtgaggaag ggtccacacc cggagtgttg aacccagctt tcttgtacaa
     5821 agtggtgtaa gcggccgcca gctttctaga ccagtttaaa catcaccatc accatcacta
     5881 agtgattaac ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     5941 ctggctcaca aataccactg agatcgatct ttttccctct gccaaaaatt atggggacat
     6001 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     6061 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     6121 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgtaactagc
     6181 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag catgcggagg
     6241 aaattctcct tgaagtttcc ctggtgttca aagtaaagga gtttgcacca gacgcacctc
     6301 tgttcactgg tccggcgtat taaaacacga tacattgtta ttagtacatt tattaagcgc
     6361 tagattctgt gcgttgttga tttacagaca attgttgtac gtattttaat aattcattaa
     6421 atttataatc tttagggtgg tatgttagag cgaaaatcaa atgattttca gcgtctttat
     6481 atctgaattt aaatattaaa tcctcaatag atttgtaaaa taggtttcga ttagtttcaa
     6541 acaagggttg tttttccgaa ccgatggctg gactatctaa tggattttcg ctcaacgcca
     6601 caaaacttgc caaatcttgt agcagcaatc tagctttgtc gatattcgtt tgtgttttgt
     6661 tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac
     6721 tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa tagagcttgg acatatttaa
     6781 catcgggcgt gttagcttta ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag
     6841 aagttgcttc cgaagacgat tttgccatag ccacacgacg cctattaatt gtgtcggcta
     6901 acacgtccgc gatcaaattt gtagttgagc tttttggaat tgcgatcgca taacttcgta
     6961 tagcatacat tatacgaagt tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc
     7021 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     7081 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     7141 tgctggcgtt tttccatagg ctccgccccc tgacgagcat cacaaaaatc gacgctcaag
     7201 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     7261 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     7321 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     7381 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     7441 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7501 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7561 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7621 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7681 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     7741 agatcctttg ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7801 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7861 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7921 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7981 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     8041 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     8101 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     8161 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     8221 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     8281 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8341 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8401 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8461 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8521 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8581 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8641 tttatcaggg ttattgtctc atgtccgcgc gtt
//
