LOCUS       sequence-328440-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus ORF3b 
            with N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6147)
  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_ORF3b
FEATURES             Location/Qualifiers
     source          1..6147
                     /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(3276..3300)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3335..3352
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3442..3461)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3507..3562
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3561..3580)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3650..3697
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3710..4415
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3798..4170
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3827..4415)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4424..4457)
                     /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(4625..5212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4704..4723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5225..6016,6017..6085))
                     /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     5848..5867
                     /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 aaaagttggc atggcctact gctggcgctg
     3121 caccagctgc tgcttctccg agaggttcca gaaccacaac ccccagaagg aaatggctac
     3181 ctctaccctg cagggttgtt ctctgtgcct gcagctggct gtggtggtgt gcaacagcct
     3241 gctgaccccc ttcgctagat gctgctggcc ttaataccca actttcttgt acaaagtggt
     3301 gtaagcggcc gccagctttc tagaccagtt taaacatcac catcaccatc actaagtgat
     3361 taacctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     3421 cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg acatcatgaa
     3481 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     3541 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     3601 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac tagcataacc
     3661 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgcg gaggaaattc
     3721 tccttgaagt ttccctggtg ttcaaagtaa aggagtttgc accagacgca cctctgttca
     3781 ctggtccggc gtattaaaac acgatacatt gttattagta catttattaa gcgctagatt
     3841 ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca ttaaatttat
     3901 aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct ttatatctga
     3961 atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt tcaaacaagg
     4021 gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac gccacaaaac
     4081 ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt ttgttttgta
     4141 ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca tcactgtcgt
     4201 tagtgtacaa ttgactcgac gtaaacacgt taaatagagc ttggacatat ttaacatcgg
     4261 gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg ttagaagttg
     4321 cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg gctaacacgt
     4381 ccgcgatcaa atttgtagtt gagctttttg gaattgcgat cgcataactt cgtatagcat
     4441 acattatacg aagttataag ctcggaacgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4801 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5041 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5221 tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5281 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5341 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5401 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5461 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5521 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5581 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5641 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5701 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5761 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5821 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5881 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5941 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6001 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6061 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6121 agggttattg tctcatgtcc gcgcgtt
//
