LOCUS       sequence-328445-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus ORF9bwith
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6273)
  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_ORF9b
FEATURES             Location/Qualifiers
     source          1..6273
                     /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(3402..3426)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3461..3478
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3568..3587)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3633..3688
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3687..3706)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3776..3823
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3836..4541
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3924..4296
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3953..4541)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4550..4583)
                     /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(4751..5338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4830..4849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5351..6142,6143..6211))
                     /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     5974..5993
                     /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 ttaatggatc caaaaatctc
     3121 tgaaatgcat ccagctttaa gattagttga tccacaaatt caactggctg ttactagaat
     3181 ggaaaatgct gttggtagag atcaaaataa tgttggtcca aaagtttacc caattatttt
     3241 aagactgggt tctccattat ctttgaatat ggctagaaaa actctgaatt ctttggaaga
     3301 taaggctttt caattgactc caattgctgt tcaaatgaca aaattagcta caacagaaga
     3361 attgccagat gaatttgttg ttgttacagt taaataataa aacccagctt tcttgtacaa
     3421 agtggtgtaa gcggccgcca gctttctaga ccagtttaaa catcaccatc accatcacta
     3481 agtgattaac ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     3541 ctggctcaca aataccactg agatcgatct ttttccctct gccaaaaatt atggggacat
     3601 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     3661 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     3721 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgtaactagc
     3781 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag catgcggagg
     3841 aaattctcct tgaagtttcc ctggtgttca aagtaaagga gtttgcacca gacgcacctc
     3901 tgttcactgg tccggcgtat taaaacacga tacattgtta ttagtacatt tattaagcgc
     3961 tagattctgt gcgttgttga tttacagaca attgttgtac gtattttaat aattcattaa
     4021 atttataatc tttagggtgg tatgttagag cgaaaatcaa atgattttca gcgtctttat
     4081 atctgaattt aaatattaaa tcctcaatag atttgtaaaa taggtttcga ttagtttcaa
     4141 acaagggttg tttttccgaa ccgatggctg gactatctaa tggattttcg ctcaacgcca
     4201 caaaacttgc caaatcttgt agcagcaatc tagctttgtc gatattcgtt tgtgttttgt
     4261 tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac
     4321 tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa tagagcttgg acatatttaa
     4381 catcgggcgt gttagcttta ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag
     4441 aagttgcttc cgaagacgat tttgccatag ccacacgacg cctattaatt gtgtcggcta
     4501 acacgtccgc gatcaaattt gtagttgagc tttttggaat tgcgatcgca taacttcgta
     4561 tagcatacat tatacgaagt tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc
     4621 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4681 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4741 tgctggcgtt tttccatagg ctccgccccc tgacgagcat cacaaaaatc gacgctcaag
     4801 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4861 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4921 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     4981 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5041 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5101 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5161 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5221 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5281 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5341 agatcctttg ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5401 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5461 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5521 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5581 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5641 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5701 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5761 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5821 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5881 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5941 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6001 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6061 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6121 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6181 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6241 tttatcaggg ttattgtctc atgtccgcgc gtt
//
