LOCUS       sequence-256911-        5712 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KSHV ORF66 a.a. 1-200 with an N-terminal 2xStrep tag in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5712)
  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 5712)
  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-2xStrep-ORF66 1-200
FEATURES             Location/Qualifiers
     source          1..5712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    819..837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     868..892
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             990..1013
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1050..1073
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1719..1736)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1725..1949
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1995..2423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2082..2101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2292..2313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2432..2452)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2437..2766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2617..2752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2679..2698
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2814..2861
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2880..3254
                     /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    3384..3505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3421..3440)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3475..3494
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3554..3570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3554..3570)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3567..3589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3578..3594
                     /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(3602..3608,3609..3626,3627..3632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3647..3668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3785..3802)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3956..4544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4036..4055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4715..5506,5507..5575))
                     /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     5338..5357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5576..5680)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
      841 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
      901 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
      961 tctagcgttt aaacttaagc ttaccaatgt ggagccaccc ccagttcgag aagggcggcg
     1021 gcagcggcgg cggcagcggc ggcggcagct ggagccaccc ccagttcgag aagggggcgg
     1081 ccgccctgga tcagcgctgg gatcgctttc ttgtgtcttg gtttggtttg gatgaggccc
     1141 agctgacggc ccaccgcgtg ttcgagggcg agaatggcgt gccagtggag gaatatgtgg
     1201 cttttgtcat attcggggag cggggtttcc agggaaacat gccctcctgg gccaggcacc
     1261 tgttggaccg cccgtcgctg gcgcaggcga tcgccgtgtt gcgcgctggc tcagacaccg
     1321 tcgccaaaca ggcgcagatt tgtgcggcgc agcagctgct tggcgcccac gtatgggttg
     1381 tggtgacgct gtcccgtgcc caggccgccg accatgctag ggcgataccg cgccacgtat
     1441 gggcaaaata cctgtcgctt ccattttcca aggcctgtgc ccaactctgc aagctgttgg
     1501 cgctttgctc tcgctttccg ttagtgacct gctgttcgaa gcccccaccg tcgctaccct
     1561 ggctaaggaa gaagtggcat gggccgttgc cgcgtcgtcc cctcctggag gtgccctcgc
     1621 ccacccgtcg cggtgtggct gcgaccggag acggtaacgg gctcgggata ggggcggcat
     1681 aatcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     1741 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1801 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1861 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1921 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1981 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2041 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2101 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2161 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2221 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2281 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2341 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2401 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2461 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2521 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2581 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2641 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2701 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2761 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
     2821 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     2881 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     2941 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     3001 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     3061 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     3121 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     3181 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     3241 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     3301 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3361 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3421 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3481 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     3541 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3601 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3661 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3721 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3781 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3841 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3901 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3961 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4021 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4081 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4141 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4201 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4261 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4321 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4381 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4441 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4501 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4561 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4621 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4681 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4741 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4801 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4861 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4921 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4981 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5041 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5101 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5161 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5221 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5281 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5341 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5401 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5461 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5521 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5581 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5641 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5701 cacctgacgt cg
//
