LOCUS       sequence-328441-        6165 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 6165)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6165)
  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..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     184..1011
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             184..308
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(346..951)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1143..1446
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1452..1729
                     /label=chicken beta-actin promoter
     primer_bind     2008..2030
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2092..2111
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2312..2335
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2342..3052
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2342..3052
                     /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(2381..2402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2642..2661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2989..3010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3053..3076
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3077..3101
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3294..3318)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3353..3370
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3460..3479)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3525..3580
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3579..3598)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3668..3715
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3728..4433
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3816..4188
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3845..4433)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4442..4475)
                     /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(4643..5230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4722..4741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5243..6034,6035..6103))
                     /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     5866..5885
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctctcctgca tcttttaatc aaatcccaag atgtgtataa acgcgccggt atgtacagga
       61 agaggtttat actaaactgt tacattgcaa acgtggtttc gtgtgccaag tgtgaaaacc
      121 gatgtttaat caaggctctg acgcatttct acaaccacga ctccaagtgt gtgggtgaag
      181 tcatgcatct tttaatcaaa tcccaagatg tgtataaacc accaaactgc caaaaaatga
      241 aaactgtcga caagctctgt ccgtttgctg gcaactgcaa gggtctcaat cctatttgta
      301 attattgaat aataaaacaa ttataaatgt caaatttgtt ttttattaac gatacaaacc
      361 aaacgcaaca agaacatttg tagtattatc tataattgaa aacgcgtagt tataatcgct
      421 gaggtaatat ttaaaatcat tttcaaatga ttcacagtta atttgcgaca atataatttt
      481 attttcacat aaactagacg ccttgtcgtc ttcttcttcg tattccttct ctttttcatt
      541 tttctcttca taaaaattaa catagttatt atcgtatcca tatatgtatc tatcgtatag
      601 agtaaatttt ttgttgtcat aaatatatat gtctttttta atggggtgta tagtaccgct
      661 gcgcatagtt tttctgtaat ttacaacagt gctattttct ggtagttctt cggagtgtgt
      721 tgctttaatt attaaattta tataatcaat gaatttggga tcgtcggttt tgtacaatat
      781 gttgccggca tagtacgcag cttcttctag ttcaattaca ccatttttta gcagcaccgg
      841 attaacataa ctttccaaaa tgttgtacga accgttaaac aaaaacagtt cacctccctt
      901 ttctatacta ttgtctgcga gcagttgttt gttgttaaaa ataacagcca ttgtaatgag
      961 acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc tgatggccgg
     1021 ccgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc
     1081 tctagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     1141 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1201 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1261 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1321 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     1381 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1441 accatgcatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1501 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1561 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
     1621 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     1681 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1741 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1801 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctt cgggctgtaa
     1861 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     1921 gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
     1981 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2041 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2101 tggttattgt gctgtctcat cattttggca aagaattgga tcggacgtct aatacgactc
     2161 actataggga catcttaagt ttattttatt ttattttatt ttattttatt ttattttatt
     2221 ttattttatt ttattttatt taaccatgac agtaatgtat aaagtctgta aagacattaa
     2281 acacgtaagt gaaaccatgg gcacctctgg tcaccaccat caccaccatc accacggcac
     2341 aagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2401 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2461 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2521 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2581 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2641 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2701 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2761 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2821 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2881 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2941 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3001 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctataca agctggaggt
     3061 gctcttccag ggtccgacaa gtttgtacaa aaaagcaggc ttaatgttcc atttggttga
     3121 tttccaagtt acaattgctg aaattttgtt gatcatcatg agaactttca aggtttctat
     3181 ttggaatttg gattacatca tcaacttgat tatcaagaac ttgtctaagt ctttgactga
     3241 aaataagtac tctcaattgg atgaagaaca accaatggaa attgattaat aaaacccagc
     3301 tttcttgtac aaagtggtgt aagcggccgc cagctttcta gaccagttta aacatcacca
     3361 tcaccatcac taagtgatta acctcaggtg caggctgcct atcagaaggt ggtggctggt
     3421 gtggccaatg ccctggctca caaataccac tgagatcgat ctttttccct ctgccaaaaa
     3481 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     3541 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     3601 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     3661 tatgtaacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3721 agcatgcgga ggaaattctc cttgaagttt ccctggtgtt caaagtaaag gagtttgcac
     3781 cagacgcacc tctgttcact ggtccggcgt attaaaacac gatacattgt tattagtaca
     3841 tttattaagc gctagattct gtgcgttgtt gatttacaga caattgttgt acgtatttta
     3901 ataattcatt aaatttataa tctttagggt ggtatgttag agcgaaaatc aaatgatttt
     3961 cagcgtcttt atatctgaat ttaaatatta aatcctcaat agatttgtaa aataggtttc
     4021 gattagtttc aaacaagggt tgtttttccg aaccgatggc tggactatct aatggatttt
     4081 cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa tctagctttg tcgatattcg
     4141 tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa aatattatgc gcttttgtat
     4201 ttctttcatc actgtcgtta gtgtacaatt gactcgacgt aaacacgtta aatagagctt
     4261 ggacatattt aacatcgggc gtgttagctt tattaggccg attatcgtcg tcgtcccaac
     4321 cctcgtcgtt agaagttgct tccgaagacg attttgccat agccacacga cgcctattaa
     4381 ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga gctttttgga attgcgatcg
     4441 cataacttcg tatagcatac attatacgaa gttataagct cggaacgctg cgctcggtcg
     4501 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4561 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4621 aaaaggccgc gttgctggcg tttttccata ggctccgccc cctgacgagc atcacaaaaa
     4681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4801 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
     4861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5221 aggatctcaa gaagatcctt tgttaccaat gcttaatcag tgaggcacct atctcagcga
     5281 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5341 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5401 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5461 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5521 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5581 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5641 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5701 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5761 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5821 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5881 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5941 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6001 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6061 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6121 attattgaag catttatcag ggttattgtc tcatgtccgc gcgtt
//
