LOCUS       sequence-257521-        6407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KSHV ORF66 a.a. 1-429 (full length) C295A mutant 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 6407)
  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 6407)
  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 C295A-2xStrep
FEATURES             Location/Qualifiers
     source          1..6407
                     /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             2306..2329
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2366..2389
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2415..2432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2421..2645
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2691..3119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2778..2797)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2988..3009
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3128..3148)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3133..3462
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3313..3448
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3510..3557
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3576..3950
                     /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    4080..4201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4117..4136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4171..4190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4250..4266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4250..4266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4263..4285)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4274..4290
                     /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(4298..4304,4305..4322,4323..4328))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4343..4364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4481..4498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4652..5240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4732..4751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5411..6202,6203..6271))
                     /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     6034..6053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6272..6376)
                     /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 atggccctgg atcagcgctg
     1021 ggatcgcttt cttgtgtctt ggtttggttt ggatgaggcc cagctgacgg cccaccgcgt
     1081 gttcgagggc gagaatggcg tgccagtgga ggaatatgtg gcttttgtca tattcgggga
     1141 gcggggtttc cagggaaaca tgccctcctg ggccaggcac ctgttggacc gcccgtcgct
     1201 ggcgcaggcg atcgccgtgt tgcgcgctgg ctcagacacc gtcgccaaac aggcgcagat
     1261 ttgtgcggcg cagcagctgc ttggcgccca cgtatgggtt gtggtgacgc tgtcccgtgc
     1321 ccaggccgcc gaccatgcta gggcgatacc gcgccacgta tgggcaaaat acctgtcgct
     1381 tccattttcc aaggcctgtg cccaactctg caagctgttg gcgctttgct ctcgctttcc
     1441 gttagtgacc tgctgttcga agcccccacc gtcgctaccc tggctaagga agaagtggca
     1501 tgggccgttg ccgcgtcgtc ccctcctgga ggtgccctcg cccacccgtc gcggtgtggc
     1561 tgcgaccgga gacggtaacg ggctcgggat aggggcggca gataccggcc tccgggaggc
     1621 cttggagcgc gtcgccccta cggttccctg cggtaacccg ttcgacgcca tgcttgggtc
     1681 actgtgtttt ttgagcctta ttaagtcccg ccacgtagtg ctgcccgcct gcgagcagga
     1741 gggacctggt ctcgtgcgga accttggcag gaggcttctg gcatacaatg ttctctcccc
     1801 ctgcgtgtcc atacccgtca tctgttcgcg cgtggcccgc gcggcccttg ccaagcgggc
     1861 gcgttgcgct cgggcggttg tggccatgga gtgtggacac tgtcttaact ttggcagggg
     1921 caagtttcat actgtcaatt ttcctcccac caacgtgttt ttcagcaggg acaggaaaga
     1981 gaagcagttc accatctgtg cgaccacggg gaggatctac tgttcttact gtggcagcga
     2041 acatatgagg gtgtatcccc tgtgcgatat taccggacgc gggaccctag cacgcgtcgt
     2101 aatcagggct gtcctagcta acaacgcggc ccttgccatt cgagacctag atcaaactgt
     2161 cagttttgta gtgccttgcc ttgggacgcc cgactgcgag gctgccctat taaagcaccg
     2221 tgacgtgcgc ggactgcttc agctcacctc acagctgctg gagttctgtt gcgggaagtg
     2281 ttcctccctc gagggaggcg gtggatggag ccacccccag ttcgagaagg gcggcggcag
     2341 cggcggcggc agcggcggcg gcagctggag ccacccccag ttcgagaagg ggcccgttta
     2401 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2461 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2521 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2581 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2641 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2701 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2761 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2821 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2881 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2941 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3001 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3061 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3121 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3181 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3241 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3301 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3361 tgactaattt tttttattta tgcagaggtc gaggccgcct ctgcctctga gctattccag
     3421 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     3481 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg
     3541 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
     3601 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     3661 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     3721 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     3781 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     3841 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     3901 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac
     3961 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4021 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4081 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4141 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4201 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4261 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4321 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4381 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4441 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4501 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4561 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4621 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4681 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4741 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4801 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4861 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4921 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4981 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5041 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5101 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5161 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5221 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5281 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5341 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5401 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5461 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5521 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5581 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5641 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5701 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5761 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5821 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5881 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5941 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6001 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6061 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6121 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6181 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6241 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6301 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6361 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
