LOCUS       sequence-335430-        6103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus ORF6 with
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6103)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6103)
  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_ORF6
FEATURES             Location/Qualifiers
     source          1..6103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     163..990
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             163..287
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(325..930)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1122..1425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1934..1956
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2018..2037
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2076..2095
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2076..2094
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2238..2261
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2268..2978
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2268..2978
                     /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(2568..2587)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2915..2936
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2979..3002
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3003..3027
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3214..3238)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3273..3290
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3380..3399)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3445..3500
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3499..3518)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3588..3635
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3648..4353
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3736..4108
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3765..4353)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4362..4395)
                     /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(4563..5150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4642..4661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5163..5954,5955..6023))
                     /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     5786..5805
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatcccaaga tgtgtataaa cgcgccggta tgtacaggaa gaggtttata ctaaactgtt
       61 acattgcaaa cgtggtttcg tgtgccaagt gtgaaaaccg atgtttaatc aaggctctga
      121 cgcatttcta caaccacgac tccaagtgtg tgggtgaagt catgcatctt ttaatcaaat
      181 cccaagatgt gtataaacca ccaaactgcc aaaaaatgaa aactgtcgac aagctctgtc
      241 cgtttgctgg caactgcaag ggtctcaatc ctatttgtaa ttattgaata ataaaacaat
      301 tataaatgtc aaatttgttt tttattaacg atacaaacca aacgcaacaa gaacatttgt
      361 agtattatct ataattgaaa acgcgtagtt ataatcgctg aggtaatatt taaaatcatt
      421 ttcaaatgat tcacagttaa tttgcgacaa tataatttta ttttcacata aactagacgc
      481 cttgtcgtct tcttcttcgt attccttctc tttttcattt ttctcttcat aaaaattaac
      541 atagttatta tcgtatccat atatgtatct atcgtataga gtaaattttt tgttgtcata
      601 aatatatatg tcttttttaa tggggtgtat agtaccgctg cgcatagttt ttctgtaatt
      661 tacaacagtg ctattttctg gtagttcttc ggagtgtgtt gctttaatta ttaaatttat
      721 ataatcaatg aatttgggat cgtcggtttt gtacaatatg ttgccggcat agtacgcagc
      781 ttcttctagt tcaattacac cattttttag cagcaccgga ttaacataac tttccaaaat
      841 gttgtacgaa ccgttaaaca aaaacagttc acctcccttt tctatactat tgtctgcgag
      901 cagttgtttg ttgttaaaaa taacagccat tgtaatgaga cgcacaaact aatatcacaa
      961 actggaaatg tctatcaata tatagttgct gatggccggc cgtaatgaga cgcacaaact
     1021 aatatcacaa actggaaatg tctatcaata tatagttgct ctagttatta atagtaatca
     1081 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1141 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1201 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1261 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1321 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1381 cctacttggc agtacatcta cgtattagtc atcgctatta ccatgcatgg tcgaggtgag
     1441 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
     1501 atttattttt taattatttt gtgcagcgat gggggcgcgg ggcgaggcgg agaggtgcgg
     1561 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     1621 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     1681 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     1741 tcccacaggt gagcgggcgg gacggccctt ctccttcggg ctgtaattag cgcttggttt
     1801 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     1861 ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1921 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     1981 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     2041 tctcatcatt ttggcaaaga attggatcgg acgtctaata cgactcacta tagggacatc
     2101 ttaagtttat tttattttat tttattttat tttattttat tttattttat tttattttat
     2161 tttatttaac catgacagta atgtataaag tctgtaaaga cattaaacac gtaagtgaaa
     2221 ccatgggcac ctctggtcac caccatcacc accatcacca cggcacaagc aagggcgagg
     2281 agctgttcac cggggtggtg cccatcctgg tcgagttgga cggcgacgta aacggccaca
     2341 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2401 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2461 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2521 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2581 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2641 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2701 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2761 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2821 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2881 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2941 ccgccgggat cactctcggc atggacgagc tatacaagct ggaggtgctc ttccagggtc
     3001 cgacaagttt gtacaaaaaa gcaggcttaa tgttccattt ggttgatttc caagttacaa
     3061 ttgctgaaat tttgttgatc atcatgagaa ctttcaaggt ttctatttgg aatttggatt
     3121 acatcatcaa cttgattatc aagaacttgt ctaagtcttt gactgaaaat aagtactctc
     3181 aattggatga agaacaacca atggaaattg ataacccagc tttcttgtac aaagtggtgt
     3241 aagcggccgc cagctttcta gaccagttta aacatcacca tcaccatcac taagtgatta
     3301 acctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     3361 caaataccac tgagatcgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     3421 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     3481 ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc
     3541 agaatgagta tttggtttag agtttggcaa catatgccca tatgtaacta gcataacccc
     3601 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa agcatgcgga ggaaattctc
     3661 cttgaagttt ccctggtgtt caaagtaaag gagtttgcac cagacgcacc tctgttcact
     3721 ggtccggcgt attaaaacac gatacattgt tattagtaca tttattaagc gctagattct
     3781 gtgcgttgtt gatttacaga caattgttgt acgtatttta ataattcatt aaatttataa
     3841 tctttagggt ggtatgttag agcgaaaatc aaatgatttt cagcgtcttt atatctgaat
     3901 ttaaatatta aatcctcaat agatttgtaa aataggtttc gattagtttc aaacaagggt
     3961 tgtttttccg aaccgatggc tggactatct aatggatttt cgctcaacgc cacaaaactt
     4021 gccaaatctt gtagcagcaa tctagctttg tcgatattcg tttgtgtttt gttttgtaat
     4081 aaaggttcga cgtcgttcaa aatattatgc gcttttgtat ttctttcatc actgtcgtta
     4141 gtgtacaatt gactcgacgt aaacacgtta aatagagctt ggacatattt aacatcgggc
     4201 gtgttagctt tattaggccg attatcgtcg tcgtcccaac cctcgtcgtt agaagttgct
     4261 tccgaagacg attttgccat agccacacga cgcctattaa ttgtgtcggc taacacgtcc
     4321 gcgatcaaat ttgtagttga gctttttgga attgcgatcg cataacttcg tatagcatac
     4381 attatacgaa gttataagct cggaacgctg cgctcggtcg ttcggctgcg gcgagcggta
     4441 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4501 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4561 tttttccata ggctccgccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4741 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4981 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5161 tgttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5221 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5281 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5341 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5401 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5461 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5521 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5581 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5641 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5701 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5761 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5821 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5881 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5941 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6001 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6061 ggttattgtc tcatgtccgc gcgtttcctg catcttttaa tca
//
