LOCUS       sequence-353292-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-tagged KSHV ORF45 RN1 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6721)
  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 6721)
  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: pDD2708
FEATURES             Location/Qualifiers
     source          1..6721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        476..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     630..650
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..743
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        724..742
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      805..814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2032..2097
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(2135..2153)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2136..2153)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2173..2190)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2179..2403
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2449..2877
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2536..2555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2746..2767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2886..2906)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2891..3220
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3071..3206
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3133..3152
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3287..4081
                     /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(3341..3360)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3951..3970
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4255..4376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4292..4311)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4346..4365
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4425..4441)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4425..4441)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4438..4460)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4449..4465
                     /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(4473..4479,4480..4497,4498..4503))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4518..4539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4656..4673)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4827..5415)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4907..4926)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5586..6377,6378..6446))
                     /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     6209..6228
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6447..6551)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6626..6645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
       61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      661 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
      721 aattaatacg actcactata gggagaccca agcttggtac cgagctcgga tccactagta
      781 acggccgcca gtgtgctgga attcgccacc atggcgatgt ttgtgaggac ctcgtctagc
      841 acacacgatg aagagagaat gcttccaatt gaaggagcgc ctcgcagacg accccccgtg
      901 aagttcatat tcccacctcc acctctttca tcacttccag gatttggcag gccgcgcggc
      961 tatgctggac ccacggtgat agatatgtct gccccagacg acgtcttcgc cgaggacacg
     1021 ccatcgccgc cagcaacccc tctggatcta cagatatccc cggatcagtc gagcggcgaa
     1081 tctgaatatg acgaggatga ggaagatgaa gatgaagaag aaaatgacga tgttcaggag
     1141 gaagacgagc cagaggggta ccctgcagac ttttttcaac ctttatctca cttgcgcccg
     1201 aggcctctgg ccagacgggc ccatacgccc aaaccggtag cagtggtagc gggccgcgtg
     1261 cgcagttcaa cggacacggc ggagtccgag gcgtccatgg gatgggttag tcaggatgac
     1321 ggattttccc ctgctgggct ctcaccttca gacgacgagg gggttgctat cctggaaccg
     1381 atggcggcat acactgggac cggggcatac ggactttcac ctgcttccag aaatagtgta
     1441 cctggaacac aaagttcacc atacagcgac cctgatgaag ggccctcgtg gcgccccctg
     1501 cgcgccgcac ccaccgcgat cgtcgacctg acatcggact ctgatagcga tgacagttcc
     1561 aactctccgg acgtgaacaa tgaggccgcg tttaccgacg cgcgccattt ttcccaccag
     1621 ccaccctcgt ccgaggagga cggagaagac caaggggaag cagcgagtca gagaatcggg
     1681 ctcatggacg tgggccagaa gcgcaaaagg cagtctaccg cctcctctgg tagcgaggat
     1741 gtggtgcgct gccagagaca accaaactta agccgcaaag cagtggcgtc cgtgataatt
     1801 atatcctcgg ggagtgacac agacgaggag ccctcgtccg ccgtgagcgt gatcgtgtct
     1861 ccgtcgagca caaagggtca cctcccaacc caatctccca gtacttccgc ccactcgatt
     1921 tcatcaggaa gcacaactac cgcggggtcc aggtgcagcg acccaacccg catcctggcc
     1981 tccacgccac ccctgtgtgg aaacggtgca tataactggc cgtggctgga cgactacaag
     2041 gaccacgacg gtgactacaa ggaccacgac atcgactaca aggacgacga cgacaagtga
     2101 gcggccgctc gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag
     2161 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2221 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2281 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2341 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2401 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2461 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2521 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2581 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2641 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2701 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2761 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2821 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2881 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2941 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3001 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3061 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3121 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3181 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3241 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3301 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3361 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3421 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3481 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3541 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3601 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3661 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3721 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3781 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3841 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3901 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3961 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4021 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4081 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4141 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4201 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4261 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4321 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4381 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4441 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4501 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4561 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4621 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4681 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4741 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4801 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4861 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4921 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4981 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5041 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5101 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5161 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5221 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5281 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5341 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5401 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5461 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5521 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5581 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5641 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5701 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5761 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5821 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5881 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5941 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6001 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6061 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6121 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6181 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6241 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6301 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6361 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6421 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6481 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6541 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     6601 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6661 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     6721 c
//
