LOCUS       sequence-336497-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus NSP7 with
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6225)
  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_NSP7
FEATURES             Location/Qualifiers
     source          1..6225
                     /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(3354..3378)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3413..3430
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3520..3539)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3585..3640
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3639..3658)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3728..3775
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3788..4493
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3876..4248
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3905..4493)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4502..4535)
                     /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(4703..5290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4782..4801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5303..6094,6095..6163))
                     /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     5926..5945
                     /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 gggggggggg gggsggggsg gggsgaggcg
     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 atgtctaaga tgtctgatgt
     3121 taagtgtact tctgttgttt tattgtctgt tttgcaacaa ttgagagttg aatcttcttc
     3181 taaattgtgg gctcaatgtg ttcaattaca taatgatatc ctgttggcta aagatacaac
     3241 tgaagctttt gaaaaaatgg tttctttgtt gtctgtctta ttgtctatgc aaggtgctgt
     3301 tgatattaat aaattgtgtg aagagatgct ggataataga gctacattgc aaaacccagc
     3361 tttcttgtac aaagtggtgt aagcggccgc cagctttcta gaccagttta aacatcacca
     3421 tcaccatcac taagtgatta acctcaggtg caggctgcct atcagaaggt ggtggctggt
     3481 gtggccaatg ccctggctca caaataccac tgagatcgat ctttttccct ctgccaaaaa
     3541 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     3601 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     3661 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     3721 tatgtaacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3781 agcatgcgga ggaaattctc cttgaagttt ccctggtgtt caaagtaaag gagtttgcac
     3841 cagacgcacc tctgttcact ggtccggcgt attaaaacac gatacattgt tattagtaca
     3901 tttattaagc gctagattct gtgcgttgtt gatttacaga caattgttgt acgtatttta
     3961 ataattcatt aaatttataa tctttagggt ggtatgttag agcgaaaatc aaatgatttt
     4021 cagcgtcttt atatctgaat ttaaatatta aatcctcaat agatttgtaa aataggtttc
     4081 gattagtttc aaacaagggt tgtttttccg aaccgatggc tggactatct aatggatttt
     4141 cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa tctagctttg tcgatattcg
     4201 tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa aatattatgc gcttttgtat
     4261 ttctttcatc actgtcgtta gtgtacaatt gactcgacgt aaacacgtta aatagagctt
     4321 ggacatattt aacatcgggc gtgttagctt tattaggccg attatcgtcg tcgtcccaac
     4381 cctcgtcgtt agaagttgct tccgaagacg attttgccat agccacacga cgcctattaa
     4441 ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga gctttttgga attgcgatcg
     4501 cataacttcg tatagcatac attatacgaa gttataagct cggaacgctg cgctcggtcg
     4561 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4621 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4681 aaaaggccgc gttgctggcg tttttccata ggctccgccc cctgacgagc atcacaaaaa
     4741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5281 aggatctcaa gaagatcctt tgttaccaat gcttaatcag tgaggcacct atctcagcga
     5341 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5401 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5461 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5521 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5581 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5641 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5701 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5761 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5821 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5881 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5941 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6001 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6061 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6121 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6181 attattgaag catttatcag ggttattgtc tcatgtccgc gcgtt
//
