LOCUS       sequence-320674-        6033 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses ORF68 K395A/K396A in mammalian cells with an N-terminal 
            TwinStrep tag and HRV 3C protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6033)
  AUTHORS   Didychuk AL, Gates SN, Gardner MR, Strong LM, Martin A, Glaunsinger 
            BA
  TITLE     A pentameric protein ring with novel architecture is required for 
            herpesviral packaging.
  JOURNAL   Elife. 2021 Feb 8;10. pii: 62261. doi: 10.7554/eLife.62261.
  PUBMED    33554858
REFERENCE   2  (bases 1 to 6033)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2021-02-8"; volume: "10. pii: 62261. doi"; pages: " 
            10.7554/eLife.62261"
COMMENT     Sequence Label: pUE1-TSP-ORF68 K395A/K396A
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(814..819,820..843,844..879,880..903)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             910..933
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      1208..1217
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2362..2934
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2529..2546)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     regulatory      2936..2945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3216..3235)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3260..3307
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(3394..3411)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3394..3410)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3403..3425)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3619..3638)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3738..3760
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3798..3816)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3884..3988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3989..4057,4058..4849)
                     /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     complement(4207..4226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5020..5608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5509..5528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5762..5779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5896..5917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5932..5937,5938..5955,5956..5962)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5970..5986
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5975..5997
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5994..6010
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5994..6010
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaattctaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctggt ttagtgaacc gaattcgtac ttcaagaact
      601 gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc
      661 tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc
      721 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
      781 aacccactgc ttaagcctcc ccgggctcga atgagtgcgt ggagtcatcc tcaattcgag
      841 aaaggtggag gttctggcgg tggatcggga ggttcagcgt ggagccaccc gcagttcgaa
      901 aaatccggac tggaagttct gttccagggg cccatgtttg ttccctggca actcggtaca
      961 attacccgtc accgagatga gctccaaaaa ctactggcag cctccctgct cccggagcac
     1021 ccggaggaga gcctcggtaa ccccataatg acacagattc accagtcgct ccaaccatct
     1081 tccccctgca gggtctgtca gctcctattt tctctggtcc gcgattcgtc cacccccatg
     1141 ggtttcttcg aggactatgc ctgcctctgc ttcttctgtc tatacgcccc acactgctgg
     1201 acctcgacca tggcggcagc ggcagacctg tgcgagatca tgcatctgca ctttccagaa
     1261 gaggaggcga catacgggct attcggaccg ggtcgcctta tgggtatcga cttgcagctg
     1321 cacttctttg ttcaaaagtg ctttaagacc accgccgccg aaaaaatact gggaatatcc
     1381 aacctgcaat ttttaaaatc agaattcatc cggggcatgc tcacaggcac catcacctgc
     1441 aacttctgct tcaaaacgtc ctggcccagg acagacaagg aggaggccac cggccccacc
     1501 ccatgctgcc agattacaga caccaccacc gcacccgcga gcggcatacc ggaactagcc
     1561 cgggccacat tctgcggcgc aagtcgcccc acaaagccca gcctacttcc cgcgctaata
     1621 gatatctggt ccacgagctc agagctcctt gacgagccgc gccctcgact gatcgcaagc
     1681 gacatgagtg aactcaaatc cgtggtcgca tcccacgatc cgttcttctc tcccccgctt
     1741 caggcagaca cctcccaggg tccatgtcta atgcacccaa ccctggggct acgatacaaa
     1801 aacgggactg catccgtctg cctcctctgc gagtgccttg cggcacaccc agaggcaccc
     1861 aaggcgctgc agacccttca gtgcgaggta atgggccata tagaaaacaa cgtaaagctg
     1921 gtagacagaa ttgcctttgt gttggacaac ccattcgcca tgccatatgt ctcagatccg
     1981 ctacttagag agctgatccg gggctgtacc ccacaggaaa ttcacaagca cctgttctgc
     2041 gacccgctgt gcgccctcaa tgctaaggtg gtgtcagagg acgtactatt ccgcctgccc
     2101 agggagcagg agtatgcggc gctcagggca tccgcggccg ccggacagct cctcgatgcc
     2161 aacaccctgt tcgactgcga ggtcgtgcag actttggtct ttctctttaa gggtctccaa
     2221 aacgccaggg tggggaaaac cacctcacta gacattattc gggagctaac cgcacaacta
     2281 aaaagacacc gcctagacct ggcccacccc tcacagacgt cacacttgta cgcttgatcg
     2341 acctgcaggc atgcaagctt gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     2401 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     2461 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     2521 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     2581 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     2641 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     2701 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     2761 cacctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     2821 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt
     2881 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatgccac
     2941 catggagcca gtagatccta gactagagcc ctggaagcat ccaggaagtc agcctaaaac
     3001 tgcttgtacc aattgctatt gtaaaaagtg ttgctttcat tgccaagttt gtttcataac
     3061 aaaagcctta ggcatctcct atggcaggaa gaagcggaga cagcgacgaa gagctcatca
     3121 gaacagtcag actcatcaag cttctctatc aaagcaaccc acctcccaac cccgagggga
     3181 cccgacaggc ccgaaggaat aggggggagg ctaactgaaa cacggaagga gacaataccg
     3241 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     3301 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     3361 ccccattggg gagcttgata tcgatagctt ggcactggcc gtcgttttac aacgtcgtga
     3421 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     3481 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     3541 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3601 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3661 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3721 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3781 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3841 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3901 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3961 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4021 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4081 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4141 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4201 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4261 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4321 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4381 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4441 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4501 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4561 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4621 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4681 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4741 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4801 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4861 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4921 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4981 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5041 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5101 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5161 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5221 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5281 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5341 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5401 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5461 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5521 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5581 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5641 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5701 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5761 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     5821 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     5881 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     5941 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6001 cagctatgac catgattacg aattatcgat atc
//
