LOCUS       sequence-336868-        7779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus NSP13 
            with N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7779)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7779)
  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_NSP13
FEATURES             Location/Qualifiers
     source          1..7779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2147..2166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2147..2165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2309..2332
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2339..3049
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2339..3049
                     /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(2639..2658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2986..3007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3050..3073
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3074..3098
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4908..4932)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4967..4984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5074..5093)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5139..5194
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5193..5212)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5282..5329
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5342..6047
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5430..5802
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5459..6047)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6056..6089)
                     /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(6257..6844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6336..6355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6857..7648,7649..7717))
                     /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     7480..7499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccgg gggggggggg ggggggsggg gggsggggsg gggsgrggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gacgtctaat acgactcact
     2161 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
     2221 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
     2281 cgtaagtgaa accatgggca cctctggtca ccaccatcac caccatcacc acggcacaag
     2341 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagttgg acggcgacgt
     2401 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2461 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2521 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2581 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2641 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2701 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2761 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2821 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2881 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2941 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3001 gttcgtgacc gccgccggga tcactctcgg catggacgag ctatacaagc tggaggtgct
     3061 cttccagggt ccgacaagtt tgtacaaaaa agcaggctta atggctgttg gtgcttgtgt
     3121 tttgtgtaat tctcaaacat ctttgagatg tggtgcttgt attagaagac catttttgtg
     3181 ttgtaagtgt tgttatgatc acgttatttc tacttctcat aagttggttt tgtctgttaa
     3241 tccatatgtt tgtaacgctc caggttgtga tgttacagat gttactcaat tatacttggg
     3301 tggtatgtct tattattgta aatctcataa gcccccaatt tcttttccat tatgtgctaa
     3361 tggtcaagtt tttggtttat ataagaacac ttgcgttggt tctgataatg ttacagattt
     3421 taacgctatc gctacatgtg attggacaaa tgctggtgat tatattttgg ctaatacatg
     3481 tactgagaga ttaaaattgt tcgctgctga aactttgaaa gctactgaag aaacatttaa
     3541 gttgtcttat ggtatcgcta cagttagaga agttttgtct gatagagaat tgcatttgtc
     3601 ttgggaagtt ggtaaaccaa gaccaccatt gaatagaaat tatgttttca caggttacag
     3661 ggttactaaa aattctaaag ttcagatcgg tgaatacaca tttgaaaaag gtgattatgg
     3721 tgatgctgtt gtttatagag gtacaactac atataagttg aacgttggtg attatttcgt
     3781 tttgacatct catacagtta tgccattatc tgctccaact ttggttccac aagaacatta
     3841 tgttagaatt acaggtttgt acccaacatt aaatatctct gatgaattct cttccaacgt
     3901 tgctaattat caaaaagttg gtatgcaaaa gtactctaca ttgcaaggtc caccaggtac
     3961 aggtaaatct cattttgcta ttggtttggc tttatattac ccatctgcta gaattgttta
     4021 cactgcttgt tctcatgctg ctgttgatgc tttgtgtgaa aaagctttga aatatttgcc
     4081 aatcgataag tgttctagaa ttattccagc tagagctaga gttgaatgtt ttgataaatt
     4141 caaggtcaac tctactttgg aacaatatgt tttttgcact gttaacgctt tgccagaaac
     4201 tacagctgat attgttgttt ttgatgaaat ctctatggct actaattacg atttgtctgt
     4261 tgttaatgct agattaagag ctaaacatta cgtttatatc ggtgatccag ctcaattacc
     4321 agctccaaga acattattaa caaaaggtac tttggagcca gaatatttta attctgtttg
     4381 tagactgatg aagacaattg gtccagatat gtttttaggt acatgtagaa gatgtccagc
     4441 tgaaattgtt gatacagttt ctgctttagt ttacgataat aagttgaagg ctcataaaga
     4501 taagtctgct caatgtttta agatgtttta caagggtgtt atcacacatg atgtttcttc
     4561 tgctattaat agaccacaaa ttggtgttgt tagagaattt ttgactagaa atctggcttg
     4621 gagaaaagct gtttttattt ctccatataa ctcccaaaac gctgttgctt ctaaaatttt
     4681 aggtttacca actcaaaccg ttgattcttc tcaaggttct gaatgtgatt atgttatttt
     4741 cacacagaca actgaaactg ctcattcttg taatgttaat agattcaacg tcgctattac
     4801 tagagctaaa gttggtattt tatgcattat gtctgacaga gatttgtatg ataagttgca
     4861 atttacctct ttggaaattc caagaagaaa tgttgctaca ttgcaaaacc cagctttctt
     4921 gtacaaagtg gtgtaagcgg ccgccagctt tctagaccag tttaaacatc accatcacca
     4981 tcactaagtg attaacctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     5041 aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca aaaattatgg
     5101 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     5161 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     5221 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgta
     5281 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaagcatg
     5341 cggaggaaat tctccttgaa gtttccctgg tgttcaaagt aaaggagttt gcaccagacg
     5401 cacctctgtt cactggtccg gcgtattaaa acacgataca ttgttattag tacatttatt
     5461 aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat tttaataatt
     5521 cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt
     5581 ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg tttcgattag
     5641 tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga ttttcgctca
     5701 acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg
     5761 ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt
     5821 catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataga gcttggacat
     5881 atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc caaccctcgt
     5941 cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta ttaattgtgt
     6001 cggctaacac gtccgcgatc aaatttgtag ttgagctttt tggaattgcg atcgcataac
     6061 ttcgtatagc atacattata cgaagttata agctcggaac gctgcgctcg gtcgttcggc
     6121 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6181 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6241 ccgcgttgct ggcgtttttc cataggctcc gccccctgac gagcatcaca aaaatcgacg
     6301 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6361 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6421 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6481 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6541 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6601 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6661 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6721 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6781 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6841 tcaagaagat cctttgttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6901 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6961 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7021 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7081 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7141 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7201 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7261 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7321 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7381 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7441 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7501 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7561 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7621 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7681 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7741 gaagcattta tcagggttat tgtctcatgt ccgcgcgtt
//
