LOCUS       sequence-256916-        5813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KSHV ORF66 a.a. 200-429 with a C-terminal 2xStrep tag in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  AUTHORS   Didychuk AL, Castaneda AF, Kushnir LO, Huang CJ, Glaunsinger BA
  TITLE     Conserved CxnC motifs in Kaposi's sarcoma-associated herpesvirus 
            ORF66 are required for viral late gene expression and are essential 
            for its interaction with ORF34.
  JOURNAL   J Virol. 2019 Oct 2. pii: JVI.01299-19. doi: 10.1128/JVI.01299-19.
  PUBMED    31578296
REFERENCE   2  (bases 1 to 5813)
  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"; journalName: "J Virol. 
            2019 Oct 2. pii: JVI.01299-19. doi: 10.1128/JVI.01299-19."
COMMENT     Sequence Label: pcDNA4/TO-ORF66 200-429-2xStrep
FEATURES             Location/Qualifiers
     source          1..5813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1712..1735
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1772..1795
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1821..1838)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1827..2051
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2097..2525
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2184..2203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2394..2415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2534..2554)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2539..2868
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2719..2854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2781..2800
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2916..2963
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2982..3356
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3486..3607
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3523..3542)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3577..3596
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3656..3672)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3656..3672)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3669..3691)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3680..3696
                     /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)."
     promoter        complement(join(3704..3710,3711..3728,3729..3734))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3749..3770
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3887..3904)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4058..4646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4138..4157)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4817..5608,5609..5677))
                     /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     5440..5459
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5678..5782)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc atggcagata ccggcctccg
     1021 ggaggccttg gagcgcgtcg cccctacggt tccctgcggt aacccgttcg acgccatgct
     1081 tgggtcactg tgttttttga gccttattaa gtcccgccac gtagtgctgc ccgcctgcga
     1141 gcaggaggga cctggtctcg tgcggaacct tggcaggagg cttctggcat acaatgttct
     1201 ctccccctgc gtgtccatac ccgtcatctg ttcgcgcgtg gcccgcgcgg cccttgccaa
     1261 gcgggcgcgt tgcgctcggg cggttgtgtg catggagtgt ggacactgtc ttaactttgg
     1321 caggggcaag tttcatactg tcaattttcc tcccaccaac gtgtttttca gcagggacag
     1381 gaaagagaag cagttcacca tctgtgcgac cacggggagg atctactgtt cttactgtgg
     1441 cagcgaacat atgagggtgt atcccctgtg cgatattacc ggacgcggga ccctagcacg
     1501 cgtcgtaatc agggctgtcc tagctaacaa cgcggccctt gccattcgag acctagatca
     1561 aactgtcagt tttgtagtgc cttgccttgg gacgcccgac tgcgaggctg ccctattaaa
     1621 gcaccgtgac gtgcgcggac tgcttcagct cacctcacag ctgctggagt tctgttgcgg
     1681 gaagtgttcc tccgtcgagg gaggcggtgg atggagccac ccccagttcg agaagggcgg
     1741 cggcagcggc ggcggcagcg gcggcggcag ctggagccac ccccagttcg agaaggggcc
     1801 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1861 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1921 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1981 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2041 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2101 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2161 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2221 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2281 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2341 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2401 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2461 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2521 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2581 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2701 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2761 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2821 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2881 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     2941 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     3001 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     3061 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     3121 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     3181 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     3241 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     3301 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     3361 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     3421 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     3481 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3541 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3601 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     3661 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3721 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3781 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3841 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3901 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3961 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4021 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4081 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4141 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4201 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4261 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4321 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4381 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4441 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4501 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4561 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4621 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4681 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4741 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4801 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4861 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4921 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4981 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5041 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5101 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5161 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5221 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5281 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5341 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5401 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5461 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5521 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5581 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5641 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5701 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5761 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
