LOCUS       sequence-345165-        6240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses FLAGHA-tagged RNAsH1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6240)
  TITLE     RNAseH1 plasmids (unpublished)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6240)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFRT-TODestFLAGHA_RNAseH1
FEATURES             Location/Qualifiers
     source          1..6240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    828..846
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    849..867
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     877..901
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1001..1024
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1025..1051
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1067..1091
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2002..2026)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2079..2120
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2200..2217)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2206..2430
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2563..2582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2714..2761
                     /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             2769..3791
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2769..3791
                     /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    3921..4042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3958..3977)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4012..4031
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4091..4107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4091..4107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4104..4126)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4115..4131
                     /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(4139..4145,4146..4163,4164..4169))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4184..4205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4322..4339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4493..5081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4573..4592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5252..6043,6044..6112))
                     /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     5875..5894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6113..6217)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
       61 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      121 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      181 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      241 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      301 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      361 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      421 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      481 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      541 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      601 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      661 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      721 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      781 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      841 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      901 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      961 cctccggact ctagcgttta aacttaagct tggtaccatg gactacaagg acgacgatga
     1021 caagtaccct tatgacgtgc ccgattacgc tttgaaccag atatcaacaa gtttgtacaa
     1081 aaaagcaggc tccaccatgg gaaccaattc agtcgacatg agctggcttc tgttcctggc
     1141 ccacagagtc gccttggccg ccttgccctg ccgccgcggc tctcgcgggt tcgggatgtt
     1201 ctatgccgtg aggaggggcc gcaagaccgg ggtctttctg acctggaatg agtgcagagc
     1261 acaggtggac cggtttcctg ctgccagatt taagaagttt gccacagagg atgaggcctg
     1321 ggcctttgtc aggaaatctg caagcccgga agtttcagaa gggcatgaaa atcaacatgg
     1381 acaagaatcg gaggcgaaag ccagcaagcg actccgtgag ccactggatg gagatggaca
     1441 tgaaagcgca gagccgtatg caaagcacat gaagccgagc gtggagccgg cgcctccagt
     1501 tagcagagac acgttttcct acatgggaga cttcgtcgtc gtctacactg atggctgctg
     1561 ctccagtaat gggcgtagaa ggccgcgagc aggaatcggc gtttactggg ggccgggcca
     1621 tcctttaaat gtaggcatta gacttcctgg gcggcagaca aaccaaagag cggaaattca
     1681 tgcagcctgc aaagccattg aacaagcaaa gactcaaaac atcaataaac tggttctgta
     1741 tacagacagt atgtttacga taaatggtat aactaactgg gttcaaggtt ggaagaaaaa
     1801 tgggtggaag acaagtgcag ggaaagaggt gatcaacaaa gaggactttg tggcactgga
     1861 gaggcttacc caggggatgg acattcagtg gatgcatgtt cctggtcatt cgggatttat
     1921 aggcaatgaa gaagctgaca gattagccag agaaggagct aaacaatcgg aagactgagc
     1981 ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggttgat atccagcaca
     2041 gtggcggccg ctcaagtcta gagggcccgc ggttcgaagg taagcctatc cctaaccctc
     2101 tcctcggtct cgattctacg cgtacctgat ccgcggccgc atagataact gatccagtgt
     2161 gcctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2221 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2281 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2341 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2401 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2461 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2521 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2581 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2641 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2701 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     2761 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     2821 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     2881 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     2941 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     3001 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     3061 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     3121 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     3181 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     3241 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     3301 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     3361 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     3421 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     3481 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3541 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     3601 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     3661 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     3721 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     3781 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3841 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3901 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3961 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4021 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4081 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4141 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4201 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4261 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4321 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4381 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4441 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4501 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4561 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4621 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4681 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4741 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4801 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4861 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4921 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4981 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5041 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5101 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5161 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5221 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5281 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5341 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5401 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5461 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5521 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5581 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5641 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5701 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5761 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5821 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5881 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5941 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6001 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6061 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
