LOCUS       sequence-308769-        6641 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cell-free/mammalian expression vector for Zika virus NS2A protein 
            with N-term mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  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 6641)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 NS2A
FEATURES             Location/Qualifiers
     source          1..6641
                     /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(3770..3794)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3829..3846
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3936..3955)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4001..4056
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4055..4074)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4144..4191
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4204..4909
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4292..4664
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4321..4909)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4918..4951)
                     /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(5119..5706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5198..5217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5719..6510,6511..6579))
                     /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     6342..6361
                     /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 gcgcgccagg gggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
     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 gggtcaaccg atcatatgga ccacttctct
     3121 cttggagtgc ttgtgattct actcatggtg caggaggggt tgaagaagag aatgaccaca
     3181 aagatcatca tgagcacatc aatggcagtg ctggtagtca tgatcttggg aggattttca
     3241 atgagtgacc tggccaagct tgtgatcctg atgggtgcta ctttcgcaga aatgaacact
     3301 ggaggagatg tagctcactt ggcattggta gcggcattta aagtcagacc agccttgctg
     3361 gtctccttca ttttcagagc caattggaca ccccgtgaga gcatgctgct agccctggct
     3421 tcgtgtcttc tgcaaactgc gatctctgct cttgaaggtg acttgatggt cctcattaat
     3481 ggatttgctt tggcctggtt ggcaattcga gcaatggccg tgccacgcac tgacaacatc
     3541 gctctaccaa tcttggctgc tctaacacca ctagctcgag gcacactgct cgtggcatgg
     3601 agagcgggcc tggctacttg tggagggatc atgctcctct ccctgaaagg gaaaggtagt
     3661 gtgaagaaga acctgccatt tgtcatggcc ctgggattga cagctgtgag ggtagtagac
     3721 cctattaatg tggtaggact actgttactc acaaggagtg ggaagcggaa cccagctttc
     3781 ttgtacaaag tggtgtaagc ggccgccagc tttctagacc agtttaaaca tcaccatcac
     3841 catcactaag tgattaacct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     3901 ccaatgccct ggctcacaaa taccactgag atcgatcttt ttccctctgc caaaaattat
     3961 ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc
     4021 attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca
     4081 aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg
     4141 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagca
     4201 tgcggaggaa attctccttg aagtttccct ggtgttcaaa gtaaaggagt ttgcaccaga
     4261 cgcacctctg ttcactggtc cggcgtatta aaacacgata cattgttatt agtacattta
     4321 ttaagcgcta gattctgtgc gttgttgatt tacagacaat tgttgtacgt attttaataa
     4381 ttcattaaat ttataatctt tagggtggta tgttagagcg aaaatcaaat gattttcagc
     4441 gtctttatat ctgaatttaa atattaaatc ctcaatagat ttgtaaaata ggtttcgatt
     4501 agtttcaaac aagggttgtt tttccgaacc gatggctgga ctatctaatg gattttcgct
     4561 caacgccaca aaacttgcca aatcttgtag cagcaatcta gctttgtcga tattcgtttg
     4621 tgttttgttt tgtaataaag gttcgacgtc gttcaaaata ttatgcgctt ttgtatttct
     4681 ttcatcactg tcgttagtgt acaattgact cgacgtaaac acgttaaata gagcttggac
     4741 atatttaaca tcgggcgtgt tagctttatt aggccgatta tcgtcgtcgt cccaaccctc
     4801 gtcgttagaa gttgcttccg aagacgattt tgccatagcc acacgacgcc tattaattgt
     4861 gtcggctaac acgtccgcga tcaaatttgt agttgagctt tttggaattg cgatcgcata
     4921 acttcgtata gcatacatta tacgaagtta taagctcgga acgctgcgct cggtcgttcg
     4981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5101 ggccgcgttg ctggcgtttt tccataggct ccgccccctg acgagcatca caaaaatcga
     5161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5521 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5701 tctcaagaag atcctttgtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5761 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5821 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5881 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5941 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6001 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6061 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6121 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6181 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6241 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6301 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6361 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6421 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6481 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6541 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6601 ttgaagcatt tatcagggtt attgtctcat gtccgcgcgt t
//
