LOCUS       sequence-353293-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-tagged KSHV ORF45 carrying 
            E223A-S226A/RC mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  AUTHORS   Alzhanova D, Meyo JO, Juarez A, Dittmer DP
  TITLE     The ORF45 Protein of Kaposi Sarcoma-Associated Herpesvirus Is an 
            Inhibitor of p53 Signaling during Viral Reactivation.
  JOURNAL   J Virol. 2021 Nov 9;95(23):e0145921. doi: 10.1128/JVI.01459-21. Epub
            2021 Sep 15.
  PUBMED    34523970
REFERENCE   2  (bases 1 to 6714)
  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."; 
            date: "2021-11-9"; volume: "95(23):e0145921. doi"; pages: " 
            10.1128/JVI.01459-21. Epub 2021 Sep 15"
COMMENT     Sequence Label: pDD2711
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        46..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     674..693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        674..692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      755..764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1982..2047
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(2078..2096)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2079..2096)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2116..2133)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2122..2346
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2392..2820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2479..2498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2689..2710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2829..2849)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2834..3163
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3014..3149
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3076..3095
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3230..4024
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3284..3303)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3894..3913
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4198..4319
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4235..4254)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4289..4308
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4368..4384)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4368..4384)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4381..4403)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4392..4408
                     /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(4416..4422,4423..4440,4441..4446))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4461..4482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4599..4616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4770..5358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4850..4869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5529..6320,6321..6389))
                     /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     6152..6171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6390..6494)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6569..6588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
       61 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      661 ggcttatcga aattaatacg actcactata gggagaccca agcttggtac cgagctcgga
      721 tccactagta acggccgcca gtgtgctgga attcgccacc atggcgatgt ttgtgaggac
      781 ctcgtctagc acacacgatg aagagagaat gcttccaatt gaaggagcgc ctcgcagacg
      841 accccccgtg aagttcatat tcccacctcc acctctttca tcacttccag gatttggcag
      901 gccgcgcggc tatgctggac ccacggtgat agatatgtct gccccagacg acgtcttcgc
      961 cgaggacacg ccatcgccgc cagcaacccc tctggatcta cagatatccc cggatcagtc
     1021 gagcggcgaa tctgaatatg acgaggatga ggaagatgaa gatgaagaag aaaatgacga
     1081 tgttcaggag gaagacgagc cagaggggta ccctgcagac ttttttcaac ctttatctca
     1141 cttgcgcccg aggcctctgg ccagacgggc ccatacgccc aaaccggtag cagtggtagc
     1201 gggccgcgtg cgcagttcaa cggacacggc ggagtccgag gcgtccatgg gatgggttag
     1261 tcaggatgac ggattttccc ctgctgggct ctcaccttca gacgacgagg gggttgctat
     1321 cctggaaccg atggcggcat acactgggac cggggcatac ggactttcac ctgcttccag
     1381 aaatagtgta cctggaacac aaagttcacc atacagcgac cctgatgctg ggcccgcgtg
     1441 gcgccccctg cgcgccgcac ccaccgcgat cgtcgacctg acatcggact ctgatagcga
     1501 tgacagttcc aactctccgg acgtgaacaa tgaggccgcg tttaccgacg cgcgccattt
     1561 ttcccaccag ccaccctcgt ccgaggagga cggagaagac caaggggaag tattgagtca
     1621 gagaatcggg ctcatggacg tgggccaggc ggccgcagcg cagtctaccg cctcctctgg
     1681 tagcgaggat gtggtgcgct gccagagaca accaaactta agccgcaaag cagtggcgtc
     1741 cgtgataatt atatcctcgg ggagtgacac agacgaggag ccctcgtccg ccgtgagcgt
     1801 gatcgtgtct ccgtcgagca caaagggtca cctcccaacc caatctccca gtacttccgc
     1861 ccactcgatt tcatcaggaa gcacaactac cgcggggtcc aggtgcagcg acccaacccg
     1921 catcctggcc tccacgccac ccctgtgtgg aaacggtgca tataactggc cgtggctgga
     1981 cgactacaag gaccacgacg gtgactacaa ggaccacgac atcgactaca aggacgacga
     2041 cgacaagtga ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta
     2101 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2161 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2221 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2281 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2341 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2401 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2461 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2521 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2581 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2641 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2701 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2761 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2821 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2881 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2941 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3001 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3061 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3121 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3181 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3241 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3301 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3361 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     3421 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3481 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3541 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3601 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3661 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3721 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     3781 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3841 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3901 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3961 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4021 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4081 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4141 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4201 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4261 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4321 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4381 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4441 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     6541 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6601 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     6661 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gtta
//
