LOCUS       sequence-335426-        6219 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 6219)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6219)
  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..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     190..1017
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             190..314
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(352..957)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1149..1452
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1969..1991
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2053..2072
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2111..2130
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2111..2129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2273..2296
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2303..3013
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2303..3013
                     /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(2603..2622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2950..2971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3014..3037
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3038..3062
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3357..3381)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3416..3433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3523..3542)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3588..3643
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3642..3661)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3731..3778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3791..4496
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3879..4251
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3908..4496)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4505..4538)
                     /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(4706..5293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4785..4804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5306..6097,6098..6166))
                     /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     5929..5948
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccgcgcgttt cctgcatctt ttaatcaaat cccaagatgt gtataaacgc gccggtatgt
       61 acaggaagag gtttatacta aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg
      121 aaaaccgatg tttaatcaag gctctgacgc atttctacaa ccacgactcc aagtgtgtgg
      181 gtgaagtcat gcatctttta atcaaatccc aagatgtgta taaaccacca aactgccaaa
      241 aaatgaaaac tgtcgacaag ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta
      301 tttgtaatta ttgaataata aaacaattat aaatgtcaaa tttgtttttt attaacgata
      361 caaaccaaac gcaacaagaa catttgtagt attatctata attgaaaacg cgtagttata
      421 atcgctgagg taatatttaa aatcattttc aaatgattca cagttaattt gcgacaatat
      481 aattttattt tcacataaac tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt
      541 ttcatttttc tcttcataaa aattaacata gttattatcg tatccatata tgtatctatc
      601 gtatagagta aattttttgt tgtcataaat atatatgtct tttttaatgg ggtgtatagt
      661 accgctgcgc atagtttttc tgtaatttac aacagtgcta ttttctggta gttcttcgga
      721 gtgtgttgct ttaattatta aatttatata atcaatgaat ttgggatcgt cggttttgta
      781 caatatgttg ccggcatagt acgcagcttc ttctagttca attacaccat tttttagcag
      841 caccggatta acataacttt ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc
      901 tcccttttct atactattgt ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt
      961 aatgagacgc acaaactaat atcacaaact ggaaatgtct atcaatatat agttgctgat
     1021 ggccggccgt aatgagacgc acaaactaat atcacaaact ggaaatgtct atcaatatat
     1081 agttgctcta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     1141 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     1201 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     1261 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1321 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1381 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1441 gctattacca tgcatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc
     1501 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg
     1561 ggcggggggg gggggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
     1621 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
     1681 gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc
     1741 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
     1801 cccttctcct tcgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg
     1861 gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg
     1921 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     1981 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     2041 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg
     2101 atcggacgtc taatacgact cactataggg acatcttaag tttattttat tttattttat
     2161 tttattttat tttattttat tttattttat tttattttat ttaaccatga cagtaatgta
     2221 taaagtctgt aaagacatta aacacgtaag tgaaaccatg ggcacctctg gtcaccacca
     2281 tcaccaccat caccacggca caagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2341 cctggtcgag ttggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2401 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2461 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2521 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2581 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2641 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2701 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2761 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2821 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2881 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2941 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3001 cgagctatac aagctggagg tgctcttcca gggtccgaca agtttgtaca aaaaagcagg
     3061 cttaatggat ccaaaaatct ctgaaatgca tccagcttta agattagttg atccacaaat
     3121 tcaactggct gttactagaa tggaaaatgc tgttggtaga gatcaaaata atgttggtcc
     3181 aaaagtttac ccaattattt taagactggg ttctccatta tctttgaata tggctagaaa
     3241 aactctgaat tctttggaag ataaggcttt tcaattgact ccaattgctg ttcaaatgac
     3301 aaaattagct acaacagaag aattgccaga tgaatttgtt gttgttacag ttaaaaaccc
     3361 agctttcttg tacaaagtgg tgtaagcggc cgccagcttt ctagaccagt ttaaacatca
     3421 ccatcaccat cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct
     3481 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa
     3541 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     3601 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     3661 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     3721 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3781 gaaagcatgc ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg
     3841 caccagacgc acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt
     3901 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt
     3961 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat
     4021 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt
     4081 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat
     4141 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat
     4201 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg
     4261 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag
     4321 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc
     4381 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat
     4441 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga
     4501 tcgcataact tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg
     4561 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4621 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa
     4741 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4801 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4861 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4921 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4981 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5041 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5101 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5161 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5221 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5281 aaaaggatct caagaagatc ctttgttacc aatgcttaat cagtgaggca cctatctcag
     5341 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5401 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5461 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5521 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5581 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5641 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5701 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5761 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5821 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5881 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5941 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6001 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6061 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6121 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6181 aatattattg aagcatttat cagggttatt gtctcatgt
//
