LOCUS       sequence-305168-        7815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS3 protein 
            with N-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7815)
  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 7815)
  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 NS3
FEATURES             Location/Qualifiers
     source          1..7815
                     /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(4944..4968)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5003..5020
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5110..5129)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5175..5230
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5229..5248)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5318..5365
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5378..6083
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5466..5838
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5495..6083)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6092..6125)
                     /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(6293..6880)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6372..6391)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6893..7684,7685..7753))
                     /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     7516..7535
                     /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 cagtggcgcc ctctgggacg tgcctgctcc
     3121 caaagaagtg aagaaaggag agaccacaga tggagtgtac agagtgatga ctcgcagact
     3181 gctaggttca acacaggttg gagtgggagt catgcaagag ggagtcttcc acaccatgtg
     3241 gcacgttaca aaaggagccg cactgaggag cggtgaggga agacttgatc catactgggg
     3301 ggatgtcaag caggacttgg tgtcatactg tgggccttgg aagttggatg cagcttggga
     3361 tggactcagc gaggtacagc ttttggccgt acctcccgga gagagggcca gaaacattca
     3421 gaccctgcct ggaatattca agacaaagga cggggacatc ggagcagttg ctctggacta
     3481 ccctgcaggg acctcaggat ctccgatcct agacaaatgt ggaagagtga taggactcta
     3541 tggcaatggg gttgtgatca agaatggaag ctatgttagt gctataaccc agggaaagag
     3601 ggaggaggag actccggttg aatgtttcga accctcgatg ctgaagaaga agcagctaac
     3661 tgtcttggat ctgcatccag gagccggaaa aaccaggaga gttcttcctg aaatagtccg
     3721 tgaagccata aaaaagagac tccggacagt gatcttggca ccaactaggg ttgtcgctgc
     3781 tgagatggag gaggccttga gaggacttcc ggtgcgttac atgacaacag cagtcaacgt
     3841 cacccattct gggacagaaa tcgttgattt gatgtgccat gccactttca cttcacgctt
     3901 actacaaccc atcagagtcc ctaattacaa tctcaacatc atggatgaag cccacttcac
     3961 agacccctca agtatagctg caagaggata catatcaaca agggttgaaa tgggcgaggc
     4021 ggctgccatt tttatgactg ccacaccacc aggaacccgt gatgcgtttc ctgactctaa
     4081 ctcaccaatc atggacacag aagtggaagt cccagagaga gcctggagct caggctttga
     4141 ttgggtgaca gaccattctg ggaaaacagt ttggttcgtt ccaagcgtga gaaacggaaa
     4201 tgaaatcgca gcctgtctga caaaggctgg aaagcgggtc atacagctca gcaggaagac
     4261 ttttgagaca gaatttcaga aaacaaaaaa tcaagagtgg gactttgtca taacaactga
     4321 catctcagag atgggcgcca acttcaaggc tgaccgggtc atagactcta ggagatgcct
     4381 aaaaccagtc atacttgatg gtgagagagt catcttggct gggcccatgc ctgtcacgca
     4441 tgctagtgct gctcagagga gaggacgtat aggcaggaac cctaacaaac ctggagatga
     4501 gtacatgtat ggaggtgggt gtgcagagac tgatgaaggc catgcacact ggcttgaagc
     4561 aagaatgctt cttgacaaca tctacctcca ggatggcctc atagcctcgc tctatcggcc
     4621 tgaggccgat aaggtagccg ccattgaggg agagtttaag ctgaggacag agcaaaggaa
     4681 gaccttcgtg gaactcatga agagaggtga ccttcccgtc tggctagcct atcaggttgc
     4741 atctgccgga ataacttaca cagacagaag atggtgcttt gatggcacaa ccaacaacac
     4801 cataatggaa gacagtgtac cagcagaggt ttggacaaag tatggagaga agagagtgct
     4861 caaaccgaga tggatggatg ctagggtctg ttcagaccat gcggccctga agtcgttcaa
     4921 agaattcgcc gctggaaaaa gaaacccagc tttcttgtac aaagtggtgt aagcggccgc
     4981 cagctttcta gaccagttta aacatcacca tcaccatcac taagtgatta acctcaggtg
     5041 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     5101 tgagatcgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     5161 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     5221 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     5281 tttggtttag agtttggcaa catatgccca tatgtaacta gcataacccc ttggggcctc
     5341 taaacgggtc ttgaggggtt ttttgctgaa agcatgcgga ggaaattctc cttgaagttt
     5401 ccctggtgtt caaagtaaag gagtttgcac cagacgcacc tctgttcact ggtccggcgt
     5461 attaaaacac gatacattgt tattagtaca tttattaagc gctagattct gtgcgttgtt
     5521 gatttacaga caattgttgt acgtatttta ataattcatt aaatttataa tctttagggt
     5581 ggtatgttag agcgaaaatc aaatgatttt cagcgtcttt atatctgaat ttaaatatta
     5641 aatcctcaat agatttgtaa aataggtttc gattagtttc aaacaagggt tgtttttccg
     5701 aaccgatggc tggactatct aatggatttt cgctcaacgc cacaaaactt gccaaatctt
     5761 gtagcagcaa tctagctttg tcgatattcg tttgtgtttt gttttgtaat aaaggttcga
     5821 cgtcgttcaa aatattatgc gcttttgtat ttctttcatc actgtcgtta gtgtacaatt
     5881 gactcgacgt aaacacgtta aatagagctt ggacatattt aacatcgggc gtgttagctt
     5941 tattaggccg attatcgtcg tcgtcccaac cctcgtcgtt agaagttgct tccgaagacg
     6001 attttgccat agccacacga cgcctattaa ttgtgtcggc taacacgtcc gcgatcaaat
     6061 ttgtagttga gctttttgga attgcgatcg cataacttcg tatagcatac attatacgaa
     6121 gttataagct cggaacgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6181 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6241 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6301 ggctccgccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6361 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6421 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6481 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6541 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6601 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6661 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6721 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6781 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6841 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgttaccaat
     6901 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6961 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7021 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7081 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7141 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7201 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7261 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7321 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7381 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7441 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7501 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7561 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7621 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7681 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7741 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7801 tcatgtccgc gcgtt
//
