LOCUS       sequence-372987-        7609 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses C terminal 3xFLAG tagged PA 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 7609)
  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 7609)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-PA-3x-FLAG-B/BRISBANE/60/2008
FEATURES             Location/Qualifiers
     source          1..7609
                     /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             3134..3199
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(3234..3252)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3235..3252)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3272..3289)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3278..3502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3548..3976
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3635..3654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3845..3866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3985..4005)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4029..4239
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4051..4247
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4098..4233
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4160..4179
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4314..5108
                     /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(4368..4387)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4978..4997
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5282..5403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5319..5338)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5373..5392
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5452..5468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5452..5468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5465..5487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5476..5492
                     /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(5500..5506,5507..5524,5525..5530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5545..5566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5683..5700)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5854..6442)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5934..5953)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6613..7404,7405..7473))
                     /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     7236..7255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7474..7578)
                     /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 ttcaccatgg atacttttat
      961 tacaagaaac ttccagacta caataataca aaaggccaaa aacacaatgg cagaatttag
     1021 tgaagatcct gaattgcaac cagcaatgct attcaatatc tgcgtccatc tagaggtttg
     1081 ctatgtaata agtgacatga attttcttga cgaagaagga aaagcatata cagcattaga
     1141 aggacaaggg aaagaacaaa acttgagacc acaatatgaa gtaattgagg gaatgccaag
     1201 aaccatagca tggatggtcc agagatcctt agctcaagag catggaatag agactcccaa
     1261 gtatctggct gatttgtttg attataaaac caaaagattt atagaagttg gaataacaaa
     1321 gggattggct gatgattact tttggaaaaa gaaagaaaag ttgggaaata gcatggaact
     1381 gatgatattc agctacaatc aagactactc gttaagtaat gaatcctcat tggatgagga
     1441 agggaaaggg agagtgctaa gcagactcac agaacttcag gctgaattaa gtctgaaaaa
     1501 tttatggcaa gttctcatag gagaagaaga tgttgaaaag ggaattgatt ttaaacttgg
     1561 acaaacaata tctagactaa gggatatatc tgttccagct ggtttctcca attttgaagg
     1621 aatgaggagc tacatagaca atatagaccc aaaaggagca atagagagaa atctagcaag
     1681 gatgtctccc ttagtatcag tcacacctaa aaagttaaca tgggaggacc taagaccaat
     1741 agggcctcac atttacgacc atgagctacc agaagttcca tataatgcct ttcttctaat
     1801 gtctgatgaa ctgggattgg ccaatatgac tgagggaaag tccaaaaaac cgaagacatt
     1861 agccaaagaa tgtctagaaa agtactcaac actacgggat caaactgacc caatattaat
     1921 aatgaaaagc gaaaaagcta acgaaaattt cctatggaag ctttggagag actgtgtaaa
     1981 tacaataagt aatgaggaaa cgagtaacga gttacagaaa accaattatg ccaaatgggc
     2041 cacaggggat ggattaacat accagaaaat aatgaaagaa gtagcaatag atgacgaaac
     2101 aatgtgccaa gaagagccta aaatccctaa caaatgtaga gtggctgctt gggttcaaac
     2161 agagatgaat ctattgagca ctctgacaag taaaagagct ctggacctac cagaaatagg
     2221 gccagacata gcacccgtgg agcatgtagg aagtgaaaga aggaaatact ttgttaatga
     2281 aatcaactac tgtaaggcct ctacagttat gatgaagtat gtgctttttc acacttcatt
     2341 gttgaatgaa agcaatgcca gcatgggaaa atacaaagta ataccaataa ccaatagagt
     2401 agtaaatgaa aaaggagaaa gtttcgacat gctttacggt ctggcggtta aaggacaatc
     2461 tcatctgagg ggagatactg atgttgtaac agttgtaact ttcgaattta gtagtacaga
     2521 tccaagagtg gactcaggaa agtggccaaa atatactgtg tttaggattg gctccctatt
     2581 tgtgagtggg agggaaaaat ctgtgtactt gtattgccga gtgaatggca caaataagat
     2641 ccaaatgaaa tggggaatgg aagctagaag atgtttgctt caatcaatgc aacaaatgga
     2701 ggcaattgtt gaacaggaat catcaataca aggatatgac atgaccaaag cctgtttcaa
     2761 gggagacaga gtaaatagcc ccaaaacttt cagtattgga actcaagaag gaaaactagt
     2821 aaaaggatcc tttggaaaag cactaagagt aatatttact aaatgcttga tgcactatgt
     2881 atttggaaat gcccaattgg aggggtttag tgccgagtct aggagacttc tactgttgat
     2941 tcaagcatta aaggacagaa agggcccttg ggtgttcgac ttagagggaa tgtattctgg
     3001 aatagaagaa tgtattagca acaacccttg ggtaatacag agtgtatact ggttcaatga
     3061 atggttgggc tttgagaagg agggcaacaa ggttctcgaa agtgtagacg agattatgga
     3121 cgaagcggcc gccgactaca aagaccatga cggtgattat aaagatcatg acatcgacta
     3181 caaggatgac gatgacaagt aggccgctcg agcatgcatc tagagggccc tattctatag
     3241 tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     3301 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3361 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3421 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3481 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     3541 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3601 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3661 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3721 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3781 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3841 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3901 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3961 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4021 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4081 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4141 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     4201 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     4261 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     4321 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4381 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4441 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4501 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4561 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4621 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4681 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4741 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4801 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4861 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4921 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4981 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5041 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5101 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     5161 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5221 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5281 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5341 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5401 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5461 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5521 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5581 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5641 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5701 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5761 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5821 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5881 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5941 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6001 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6061 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6121 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6181 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6241 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6301 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6361 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6421 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6481 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6541 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6601 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6661 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6721 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6781 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6841 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6901 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6961 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7021 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7081 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7141 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7201 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7261 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7321 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7381 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7441 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7501 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7561 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
