LOCUS       Exported                7768 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Gateway destination vector for cell-free and mamalian expression 
            with N-term 8xHis and eGFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    pmCellFree_KA1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7768)
  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 7768)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7768
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..3049
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2639..2658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2986..3007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3050..3073
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3074..3198
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3245..3550)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3347..3366)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3892..4551)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4466..4485
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4552..4654)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(4797..4921)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4956..4973
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5063..5082)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5128..5183
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5182..5201)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5271..5318
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5331..6036
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5419..5791
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5448..6036)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    6045..6078
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     rep_origin      complement(6246..6833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6325..6344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6846..7706)
                     /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     7469..7488
                     /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 gggcgaggcg
     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 gagctgttca ccggggtggt gcccatcctg gtcgagttgg acggcgacgt
     2401 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2461 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2521 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2581 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2641 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2701 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2761 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2821 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2881 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2941 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3001 gttcgtgacc gccgccggga tcactctcgg catggacgag ctatacaagc tggaggtgct
     3061 cttccagggt ccgacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     3121 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     3181 acatatccag tcactatggg gcccgtcgac ctgcagactg gctgtgtata agggagcctg
     3241 acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
     3301 tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca
     3361 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     3421 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     3481 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     3541 taaactgcat ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac
     3601 cgggcgacct cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga
     3661 cgggcttcat tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct
     3721 tgagcaactg atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc
     3781 tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc
     3841 gcgcaaatac gcatactgtt atctggcttt tagtaagccg gatccacgcg tttacgcccc
     3901 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     3961 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     4021 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     4081 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     4141 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     4201 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     4261 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     4321 cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     4381 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     4441 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4501 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4561 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4621 atggtgaaag ttggaacctc ttgcgtgccg atcaacgtct cattttcgcc aaaagttggc
     4681 ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc
     4741 cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagcttcata
     4801 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     4861 ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg
     4921 tgtaagcggc cgccagcttt ctagaccagt ttaaacatca ccatcaccat cactaagtga
     4981 ttaacctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     5041 tcacaaatac cactgagatc gatctttttc cctctgccaa aaattatggg gacatcatga
     5101 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     5161 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     5221 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgtaa ctagcataac
     5281 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt
     5341 ctccttgaag tttccctggt gttcaaagta aaggagtttg caccagacgc acctctgttc
     5401 actggtccgg cgtattaaaa cacgatacat tgttattagt acatttatta agcgctagat
     5461 tctgtgcgtt gttgatttac agacaattgt tgtacgtatt ttaataattc attaaattta
     5521 taatctttag ggtggtatgt tagagcgaaa atcaaatgat tttcagcgtc tttatatctg
     5581 aatttaaata ttaaatcctc aatagatttg taaaataggt ttcgattagt ttcaaacaag
     5641 ggttgttttt ccgaaccgat ggctggacta tctaatggat tttcgctcaa cgccacaaaa
     5701 cttgccaaat cttgtagcag caatctagct ttgtcgatat tcgtttgtgt tttgttttgt
     5761 aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg tatttctttc atcactgtcg
     5821 ttagtgtaca attgactcga cgtaaacacg ttaaatagag cttggacata tttaacatcg
     5881 ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt
     5941 gcttccgaag acgattttgc catagccaca cgacgcctat taattgtgtc ggctaacacg
     6001 tccgcgatca aatttgtagt tgagcttttt ggaattgcga tcgcataact tcgtatagca
     6061 tacattatac gaagttataa gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg
     6121 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6181 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6241 gcgtttttcc ataggctccg ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6841 ctttgttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6961 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7141 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7441 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7741 cagggttatt gtctcatgtc cgcgcgtt
//
