LOCUS       sequence-335422-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus E with 
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6163)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6163)
  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"
COMMENT     Sequence Label: pmCellFree_KA1_SARS-COV-2_E
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     159..986
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             159..283
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(321..926)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1118..1421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1948..1970
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2032..2051
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2090..2109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2090..2108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2252..2275
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2282..2992
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2282..2992
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2582..2601)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2929..2950
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2993..3016
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3017..3041
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3270..3294)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3329..3346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3436..3455)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3501..3556
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3555..3574)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3644..3691
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3704..4409
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3792..4164
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3821..4409)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4418..4451)
                     /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(4619..5206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4698..4717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5219..6010,6011..6079))
                     /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     5842..5861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccaagatgtg tataaacgcg ccggtatgta caggaagagg tttatactaa actgttacat
       61 tgcaaacgtg gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg ctctgacgca
      121 tttctacaac cacgactcca agtgtgtggg tgaagtcatg catcttttaa tcaaatccca
      181 agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtcgacaagc tctgtccgtt
      241 tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata
      301 aatgtcaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta
      361 ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca
      421 aatgattcac agttaatttg cgacaatata attttatttt cacataaact agacgccttg
      481 tcgtcttctt cttcgtattc cttctctttt tcatttttct cttcataaaa attaacatag
      541 ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata
      601 tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca
      661 acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa
      721 tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta cgcagcttct
      781 tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg
      841 tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt
      901 tgtttgttgt taaaaataac agccattgta atgagacgca caaactaata tcacaaactg
      961 gaaatgtcta tcaatatata gttgctgatg gccggccgta atgagacgca caaactaata
     1021 tcacaaactg gaaatgtcta tcaatatata gttgctctag ttattaatag taatcaatta
     1081 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1141 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1201 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1261 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1321 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1381 cttggcagta catctacgta ttagtcatcg ctattaccat gcatggtcga ggtgagcccc
     1441 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     1501 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcggg gcggggcgag
     1561 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     1621 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1681 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1741 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctt cgggctgtaa
     1801 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     1861 gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
     1921 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     1981 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2041 tggttattgt gctgtctcat cattttggca aagaattgga tcggacgtct aatacgactc
     2101 actataggga catcttaagt ttattttatt ttattttatt ttattttatt ttattttatt
     2161 ttattttatt ttattttatt taaccatgac agtaatgtat aaagtctgta aagacattaa
     2221 acacgtaagt gaaaccatgg gcacctctgg tcaccaccat caccaccatc accacggcac
     2281 aagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagt tggacggcga
     2341 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2401 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2461 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2521 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2581 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2641 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2701 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2761 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2821 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2881 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2941 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctataca agctggaggt
     3001 gctcttccag ggtccgacaa gtttgtacaa aaaagcaggc ttaatgtact ctttcgtttc
     3061 tgaagaaact ggtacattga ttgttaattc tgttttgttg ttcctggctt ttgttgtttt
     3121 tttattggtt actctggcta ttttgactgc tttaagatta tgtgcttact gttgtaatat
     3181 cgttaacgtt tctttggtta agccatcttt ttatgtttac tctagagtta agaacctgaa
     3241 ttcttctaga gttccagatt tattggttaa cccagctttc ttgtacaaag tggtgtaagc
     3301 ggccgccagc tttctagacc agtttaaaca tcaccatcac catcactaag tgattaacct
     3361 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     3421 taccactgag atcgatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     3481 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     3541 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     3601 tgagtatttg gtttagagtt tggcaacata tgcccatatg taactagcat aaccccttgg
     3661 ggcctctaaa cgggtcttga ggggtttttt gctgaaagca tgcggaggaa attctccttg
     3721 aagtttccct ggtgttcaaa gtaaaggagt ttgcaccaga cgcacctctg ttcactggtc
     3781 cggcgtatta aaacacgata cattgttatt agtacattta ttaagcgcta gattctgtgc
     3841 gttgttgatt tacagacaat tgttgtacgt attttaataa ttcattaaat ttataatctt
     3901 tagggtggta tgttagagcg aaaatcaaat gattttcagc gtctttatat ctgaatttaa
     3961 atattaaatc ctcaatagat ttgtaaaata ggtttcgatt agtttcaaac aagggttgtt
     4021 tttccgaacc gatggctgga ctatctaatg gattttcgct caacgccaca aaacttgcca
     4081 aatcttgtag cagcaatcta gctttgtcga tattcgtttg tgttttgttt tgtaataaag
     4141 gttcgacgtc gttcaaaata ttatgcgctt ttgtatttct ttcatcactg tcgttagtgt
     4201 acaattgact cgacgtaaac acgttaaata gagcttggac atatttaaca tcgggcgtgt
     4261 tagctttatt aggccgatta tcgtcgtcgt cccaaccctc gtcgttagaa gttgcttccg
     4321 aagacgattt tgccatagcc acacgacgcc tattaattgt gtcggctaac acgtccgcga
     4381 tcaaatttgt agttgagctt tttggaattg cgatcgcata acttcgtata gcatacatta
     4441 tacgaagtta taagctcgga acgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4501 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4561 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4621 tccataggct ccgccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4681 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4741 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4801 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4861 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4921 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4981 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5041 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5101 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5161 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgtt
     5221 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5281 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5341 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5401 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5461 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5521 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5581 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5641 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5701 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5761 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5821 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5881 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5941 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6001 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6061 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6121 attgtctcat gtccgcgcgt ttcctgcatc ttttaatcaa atc
//
