LOCUS       sequence-307602-        5191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tagging vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5191)
  AUTHORS   Bresson S, Robertson N, Sani E, Turowski TW, Shchepachev V, 
            Kompauerova M, Spanos C, Helwak A, Tollervey D
  TITLE     Integrative vectors for regulated expression of SARS-CoV-2 proteins 
            implicated in RNA metabolism.
  JOURNAL   Wellcome Open Res. 2020 Nov 3;5:261. doi: 
            10.12688/wellcomeopenres.16322.1. eCollection 2020.
  PUBMED    33313418
REFERENCE   2  (bases 1 to 5191)
  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: "Wellcome 
            Open Res."; date: "2020-11-3"; volume: "5:261. doi"; pages: " 
            10.12688/wellcomeopenres.16322.1. eCollection 2020"
COMMENT     Sequence Label: pcDNA5-FRT-TO-N-Flag-8His
FEATURES             Location/Qualifiers
     source          1..5191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        541..744
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     695..715
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    746..764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    767..785
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     795..819
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             916..939
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             952..975
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1069..1086)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1075..1299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1432..1451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1583..1630
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1638..2660
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1638..2660
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    2790..2911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2827..2846)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2881..2900
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2960..2976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2960..2976)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2973..2995)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2984..3000
                     /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(3008..3014,3015..3032,3033..3038))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3053..3074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3191..3208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3362..3950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3442..3461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4121..4912,4913..4981))
                     /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     4744..4763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4982..5086)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5161..5180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
       61 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      121 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      721 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      781 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      841 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      901 cttaagcttg agatggacta taaagatgac gatgacaagg ccgcagccgc acatcaccac
      961 catcatcacc atcacggatc cactagtcca gtgtggtgga attctgcaga tatccagcac
     1021 agtggcggcc gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     1081 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1141 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1201 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1261 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     1321 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1381 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1441 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1501 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1561 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta
     1621 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     1681 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     1741 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     1801 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     1861 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     1921 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     1981 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     2041 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     2101 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     2161 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     2221 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     2281 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     2341 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     2401 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     2461 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     2521 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     2581 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     2641 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg
     2701 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     2761 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     2821 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2881 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     2941 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3001 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3061 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3121 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3181 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3241 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3301 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3361 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3421 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3481 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3541 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3601 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3661 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3721 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3781 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3841 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3901 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3961 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4021 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4081 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4141 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4201 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4261 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4321 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4381 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4441 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4501 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4561 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4621 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4681 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4741 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4801 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4861 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4921 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4981 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5041 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5101 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     5161 tacaatctgc tctgatgccg catagttaag c
//
