LOCUS       sequence-372980-        7741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C terminal 3xFLAG tagged PB2 subunit of Influenza 
            B/Brisbane/60/2008 virus polymerase in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7741)
  AUTHORS   Kedia N, Banerjee S, Mondal A
  TITLE     A Comprehensive Roadmap Towards the Generation of an Influenza B 
            Reporter Assay Using a Single DNA Polymerase-Based Cloning of the 
            Reporter RNA Construct.
  JOURNAL   Front Microbiol. 2022 May 25;13:868367. doi: 
            10.3389/fmicb.2022.868367. eCollection 2022.
  PUBMED    35694292
REFERENCE   2  (bases 1 to 7741)
  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: "Front 
            Microbiol."; date: "2022-05-25"; volume: "13:868367. doi"; pages: " 
            10.3389/fmicb.2022.868367. eCollection 2022"
COMMENT     Sequence Label: pcDNA3-PB2-3x-FLAG-B/BRISBANE/60/2008
FEATURES             Location/Qualifiers
     source          1..7741
                     /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"
     CDS             3266..3331
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(3366..3384)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3367..3384)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3404..3421)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3410..3634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3680..4108
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3767..3786)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3977..3998
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4117..4137)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4161..4371
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4183..4379
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4230..4365
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4292..4311
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4446..5240
                     /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(4500..4519)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5110..5129
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5414..5535
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5451..5470)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5505..5524
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5584..5600)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5584..5600)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5597..5619)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5608..5624
                     /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(5632..5638,5639..5656,5657..5662))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5677..5698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5815..5832)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5986..6574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6066..6085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6745..7536,7537..7605))
                     /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     7368..7387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7606..7710)
                     /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 ttcaccatga cattggccaa
      961 aattgaattg ttaaaacaac tgctaaggga caatgaagcc aaaacagttt tgaagcaaac
     1021 aacggtagac caatataaca taataagaaa attcaataca tcaaggattg aaaagaatcc
     1081 ttcactaagg atgaagtggg ccatgtgttc taattttccc ttggctctaa ccaagggcga
     1141 tatggcaaac agaatcccct tggaatacaa agggatacaa cttaaaacaa atgctgaaga
     1201 cataggaacc aaaggccaaa tgtgctcaat agcagcagtt acttggtgga atacatatgg
     1261 accaatagga gatactgaag gtttcgaaag ggtctacgaa agcttttttc tcagaaaaat
     1321 gagacttgac aacgccactt ggggccgaat aacttttggc ccagttgaaa gagtgagaaa
     1381 aagggtactg ctaaaccctc tcaccaagga aatgcctccg gatgaggcga gcaatgtgat
     1441 aatggaaata ttgttcccta aagaagcagg aataccaaga gaatccactt ggatacatag
     1501 ggaactgata aaagaaaaaa gagaaaaatt gaaaggaaca atgataactc caatcgtact
     1561 ggcatacatg cttgaaagag aactggttgc tcgaagaaga ttcttgccag tggcaggagc
     1621 aacatcagct gagttcatag aaatgctaca ctgcttacaa ggtgaaaatt ggagacaaat
     1681 atatcaccca ggagggaata aattaactga gtccaggtct caatcaatga tagtagcttg
     1741 tagaaaaata atcagaagat caatagtcgc ttcaaaccca ctggagctag ctgtagaaat
     1801 tgcaaacaag actgtgatag atactgaacc tttaaagtca tgtctggcag ccatagacgg
     1861 aggtgatgta gcttgtgaca taataagagc tgcattagga ctaaagatca gacaaagaca
     1921 aagatttgga cggcttgagc taaaaagaat atcaggaaga ggattcaaaa atgatgaaga
     1981 aatattaata gggaacggaa caatacagaa gattggaata tgggacgggg aagaggagtt
     2041 ccatgtaaga tgtggtgaat gcaggggaat attaaaaaag agtaaaatga aactggaaaa
     2101 actactgata aattcagcca aaaaggagga tatgagagat ttaataatct tatgcatggt
     2161 attttctcaa gacactagga tgttccaagg agtgagagga gaaataaatt ttcttaatcg
     2221 agcaggccaa cttttatctc caatgtacca actccaacga tattttttga atagaagcaa
     2281 cgaccttttt gatcaatggg ggtatgagga atcacccaaa gcaagtgaac tacatgggat
     2341 aaatgaatca atgaatgcat ctgactatac attgaaaggg attgtagtga caagaaatgt
     2401 aattgacgac tttagctcta ttgaaacaga aaaagtatcc ataacaaaaa atcttagttt
     2461 aataaaaagg actggggaag tcataatggg agctaatgac gtgagtgaat tagaatcaca
     2521 agcacagctg atgataacat atgatacacc taaaatgtgg gaaatgggaa caaccaaaga
     2581 actggtgcaa aacacttatc aatgggtgct aaaaaacttg gtgacactga aggctcagtt
     2641 tcttctagga aaagaggaca tgttccaatg ggatgcattt gaagcatttg agagcataat
     2701 tcctcagaag atggctggtc agtacagtgg atttgcaaga gcagtgctca aacaaatgag
     2761 agaccaggag gttatgaaaa ctgaccagtt cataaagttg ttgccttttt gtttctcacc
     2821 accaaaatta aggagcaatg gggagcctta tcaattctta aaacttgtgt tgaaaggagg
     2881 aggggaaaat ttcatcgaag taaggaaagg gtcccctcta ttttcctata atccacaaac
     2941 agaagtccta actatatgcg gcagaatgat gtcattaaaa gggaaaattg aagatgaaga
     3001 aaggaataga tcaatgggta atgcagtatt agcaggcttt ctcgttagtg gcaagtatga
     3061 cccagatctt ggagatttca aaactattga agaacttgaa aagctgaaac cgggggaaaa
     3121 ggcaaacatc ttactttatc aaggaaaacc agttaaagta gttaaaagga aaaggtatag
     3181 tgctttgtcc aatgacattt cacaaggaat taagagacaa agaatgacag ttgagtctat
     3241 ggggtgggcc ttgagcgcgg ccgccgacta caaagaccat gacggtgatt ataaagatca
     3301 tgacatcgac tacaaggatg acgatgacaa gtaggccgct cgagcatgca tctagagggc
     3361 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     3421 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3481 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3541 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3601 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3661 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3721 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3781 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3841 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3901 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3961 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4021 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4081 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     4141 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4201 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4261 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4321 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4381 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     4441 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4501 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4561 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4621 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4681 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4741 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4801 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4861 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4921 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4981 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5041 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5101 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     5161 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5221 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5281 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5341 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5401 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     5461 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5521 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5581 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5641 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5701 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5761 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5821 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5881 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5941 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6001 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6061 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6121 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6181 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6241 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6301 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6361 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6421 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6481 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6541 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6601 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6661 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6721 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6781 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6841 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6901 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6961 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7021 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7081 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7141 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7201 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7261 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7321 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7381 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7441 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7501 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7561 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7621 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7681 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7741 c
//
