LOCUS       sequence-305166-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS1 protein 
            with N-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  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 7019)
  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 NS1
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     180..1007
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             180..304
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(342..947)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1139..1442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1448..1725
                     /label=chicken beta-actin promoter
     primer_bind     2004..2026
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2088..2107
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2146..2165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2146..2164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2308..2331
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2338..3039
                     /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(2477..2495)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2745..2764
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3040..3063
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3064..3088
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4148..4172)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4207..4224
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4314..4333)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4379..4434
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4433..4452)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4522..4569
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4582..5287
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4670..5042
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4699..5287)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5296..5329)
                     /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(5497..6084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5576..5595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6097..6888,6889..6957))
                     /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     6720..6739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cctgcatctt ttaatcaaat cccaagatgt gtataaacgc gccggtatgt acaggaagag
       61 gtttatacta aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg
      121 tttaatcaag gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat
      181 gcatctttta atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac
      241 tgtcgacaag ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta
      301 ttgaataata aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac
      361 gcaacaagaa catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg
      421 taatatttaa aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt
      481 tcacataaac tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc
      541 tcttcataaa aattaacata gttattatcg tatccatata tgtatctatc gtatagagta
      601 aattttttgt tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc
      661 atagtttttc tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct
      721 ttaattatta aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg
      781 ccggcatagt acgcagcttc ttctagttca attacaccat tttttagcag caccggatta
      841 acataacttt ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct
      901 atactattgt ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc
      961 acaaactaat atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccgt
     1021 aatgagacgc acaaactaat atcacaaact ggaaatgtct atcaatatat agttgctcta
     1081 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     1141 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     1201 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     1261 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     1321 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1381 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1441 tgcatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     1501 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     1561 gggggggggc gcgcgccggg gggggggggg gggggggggg gggggggggg gggggggcgg
     1621 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     1681 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
     1741 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1801 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctccttcggg ctgtaattag
     1861 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
     1921 cgggagggcc ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct
     1981 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     2041 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     2101 tattgtgctg tctcatcatt ttggcaaaga attggatcgg acgtctaata cgactcacta
     2161 tagggacatc ttaagtttat tttattttat tttattttat tttattttat tttattttat
     2221 tttattttat tttatttaac catgacagta atgtataaag tctgtaaaga cattaaacac
     2281 gtaagtgaaa ccatgggcac ctctggtcac caccatcacc accatcacca cggcacaagc
     2341 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     2401 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2461 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     2521 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     2581 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     2641 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     2701 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     2761 aagactatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     2821 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     2881 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     2941 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     3001 ggccgccact ccaccggcgg catggacgag ctgtacaagc tggaggtgct cttccagggt
     3061 ccgacaagtt tgtacaaaaa agcaggcttc gacgtggggt gctcagtgga cttctcaaaa
     3121 aaggaaacga gatgtggcac gggggtattc atctataatg atgttgaagc ctggagggac
     3181 cggtacaagt accatcctga ctccccccgc agattggcag cagcagtcaa gcaggcctgg
     3241 gaagagggga tctgtgggat ctcatccgtt tcaagaatgg aaaacatcat gtggaaatca
     3301 gtagaagggg agctcaatgc tatcctagag gagaatggag ttcaactgac agttgttgtg
     3361 ggatctgtaa aaaaccccat gtggagaggt ccacaaagat tgccagtgcc tgtgaatgag
     3421 ctgccccacg gctggaaagc ctgggggaaa tcgtattttg ttagggcggc aaagaccaac
     3481 aacagttttg ttgtcgacgg tgacacactg aaggaatgtc cgcttgagca cagagcatgg
     3541 aatagttttc ttgtggagga tcacgggttt ggagtcttcc acaccagtgt ctggcttaag
     3601 gtcagagaag attactcatt agaatgtgac ccagccgtca taggaacagc tgttaaggga
     3661 agggaggccg cgcacagtga tctgggctat tggattgaaa gtgaaaagaa tgacacatgg
     3721 aggctgaaga gggcccacct gattgagatg aaaacatgtg aatggccaaa gtctcacaca
     3781 ttgtggacag atggagtaga agaaagtgat cttatcatac ccaagtcttt agctggtcca
     3841 ctcagccacc acaacaccag agagggttac agaacccaag tgaaagggcc ttggcacagt
     3901 gaagagcttg aaatccggtt tgaggaatgt ccaggcacca aggtttacgt ggaggagaca
     3961 tgcggaacta gaggaccatc tctgagatca actactgcaa gtggaagggt cattgaggaa
     4021 tggtgctgta gggaatgcac aatgccccca ctatcgtttc gagcaaaaga cggctgctgg
     4081 tatggaatgg agataaggcc caggaaagaa ccagagagca acttagtgag gtcaatggtg
     4141 acagcgaacc cagctttctt gtacaaagtg gtgtaagcgg ccgccagctt tctagaccag
     4201 tttaaacatc accatcacca tcactaagtg attaacctca ggtgcaggct gcctatcaga
     4261 aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat cgatcttttt
     4321 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     4381 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     4441 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     4501 gcaacatatg cccatatgta actagcataa ccccttgggg cctctaaacg ggtcttgagg
     4561 ggttttttgc tgaaagcatg cggaggaaat tctccttgaa gtttccctgg tgttcaaagt
     4621 aaaggagttt gcaccagacg cacctctgtt cactggtccg gcgtattaaa acacgataca
     4681 ttgttattag tacatttatt aagcgctaga ttctgtgcgt tgttgattta cagacaattg
     4741 ttgtacgtat tttaataatt cattaaattt ataatcttta gggtggtatg ttagagcgaa
     4801 aatcaaatga ttttcagcgt ctttatatct gaatttaaat attaaatcct caatagattt
     4861 gtaaaatagg tttcgattag tttcaaacaa gggttgtttt tccgaaccga tggctggact
     4921 atctaatgga ttttcgctca acgccacaaa acttgccaaa tcttgtagca gcaatctagc
     4981 tttgtcgata ttcgtttgtg ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt
     5041 atgcgctttt gtatttcttt catcactgtc gttagtgtac aattgactcg acgtaaacac
     5101 gttaaataga gcttggacat atttaacatc gggcgtgtta gctttattag gccgattatc
     5161 gtcgtcgtcc caaccctcgt cgttagaagt tgcttccgaa gacgattttg ccatagccac
     5221 acgacgccta ttaattgtgt cggctaacac gtccgcgatc aaatttgtag ttgagctttt
     5281 tggaattgcg atcgcataac ttcgtatagc atacattata cgaagttata agctcggaac
     5341 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5401 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5461 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccccctgac
     5521 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5581 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5641 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5701 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5761 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5821 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5881 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5941 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6001 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6061 acgcgcagaa aaaaaggatc tcaagaagat cctttgttac caatgcttaa tcagtgaggc
     6121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6181 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6241 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6301 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6361 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6421 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6481 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6541 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6601 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6661 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6721 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6781 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6841 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6901 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6961 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatgt ccgcgcgtt
//
