LOCUS       sequence-345719-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-tagged KSHV ORF45 E223A-S226A 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: pDD2703
FEATURES             Location/Qualifiers
     source          1..6721
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      944..953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2171..2236
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(2274..2292)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2275..2292)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2312..2329)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2318..2542
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2588..3016
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2675..2694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2885..2906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3025..3045)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3030..3359
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3210..3345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3272..3291
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3426..4220
                     /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(3480..3499)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4090..4109
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4394..4515
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4431..4450)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4485..4504
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4564..4580)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4564..4580)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4577..4599)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4588..4604
                     /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(4612..4618,4619..4636,4637..4642))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4657..4678
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4795..4812)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4966..5554)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5046..5065)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5725..6516,6517..6585))
                     /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     6348..6367
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6586..6690)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgccacca tggcgatgtt
      961 tgtgaggacc tcgtctagca cacacgatga agagagaatg cttccaattg aaggagcgcc
     1021 tcgcagacga ccccccgtga agttcatatt cccacctcca cctctttcat cacttccagg
     1081 atttggcagg ccgcgcggct atgctggacc cacggtgata gatatgtctg ccccagacga
     1141 cgtcttcgcc gaggacacgc catcgccgcc agcaacccct ctggatctac agatatcccc
     1201 ggatcagtcg agcggcgaat ctgaatatga cgaggatgag gaagatgaag atgaagaaga
     1261 aaatgacgat gttcaggagg aagacgagcc agaggggtac cctgcagact tttttcaacc
     1321 tttatctcac ttgcgcccga ggcctctggc cagacgggcc catacgccca aaccggtagc
     1381 agtggtagcg ggccgcgtgc gcagttcaac ggacacggcg gagtccgagg cgtccatggg
     1441 atgggttagt caggatgacg gattttcccc tgctgggctc tcaccttcag acgacgaggg
     1501 ggttgctatc ctggaaccga tggcggcata cactgggacc ggggcatacg gactttcacc
     1561 tgcttccaga aatagtgtac ctggaacaca aagttcacca tacagcgacc ctgatgctgg
     1621 gcccgcgtgg cgccccctgc gcgccgcacc caccgcgatc gtcgacctga catcggactc
     1681 tgatagcgat gacagttcca actctccgga cgtgaacaat gaggccgcgt ttaccgacgc
     1741 gcgccatttt tcccaccagc caccctcgtc cgaggaggac ggagaagacc aaggggaagt
     1801 attgagtcag agaatcgggc tcatggacgt gggccagaag cgcaaaaggc agtctaccgc
     1861 ctcctctggt agcgaggatg tggtgcgctg ccagagacaa ccaaacttaa gccgcaaagc
     1921 agtggcgtcc gtgataatta tatcctcggg gagtgacaca gacgaggagc cctcgtccgc
     1981 cgtgagcgtg atcgtgtctc cgtcgagcac aaagggtcac ctcccaaccc aatctcccag
     2041 tacttccgcc cactcgattt catcaggaag cacaactacc gcggggtcca ggtgcagcga
     2101 cccaacccgc atcctggcct ccacgccacc cctgtgtgga aacggtgcat ataactggcc
     2161 gtggctggac gactacaagg accacgacgg tgactacaag gaccacgaca tcgactacaa
     2221 ggacgacgac gacaagtgag cggccgctcg agcatgcatc tagagggccc tattctatag
     2281 tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     2341 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2401 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2461 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2521 ggggatgcgg tgggctctat ggcttctgag gcagaaagaa ccagctgggg ctctaggggg
     2581 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2641 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2701 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2761 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2821 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2881 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2941 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3001 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3061 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3121 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3181 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3241 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3301 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3361 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3421 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3481 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3541 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3601 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3661 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3721 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3781 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3841 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3901 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3961 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4021 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4081 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4141 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4201 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4261 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4321 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4381 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4441 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4501 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4561 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4621 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4681 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4741 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4801 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4861 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4921 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4981 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5041 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5101 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5161 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5221 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5281 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5341 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5401 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5461 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5521 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5581 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5641 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5701 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5761 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5821 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5881 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5941 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6001 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6061 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6121 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6181 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6241 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6301 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6361 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6421 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6481 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6541 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6601 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6661 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6721 c
//
