LOCUS       sequence-354766-        7668 bp    DNA     circular SYN 14-APR-2025
DEFINITION  expresses RFP-tagged RNAseH1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7668)
  TITLE     RNAseH1 plasmids (unpublished)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7668)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFRT-TODestRFP_RNAseH1
FEATURES             Location/Qualifiers
     source          1..7668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             862..2289
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     protein_bind    complement(3230..3254)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3307..3348
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3386..3403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3392..3616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3749..3768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3900..3947
                     /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             3955..4977
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3955..4977
                     /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    5107..5228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5144..5163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5198..5217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5277..5293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5277..5293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5290..5312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5301..5317
                     /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(5325..5331,5332..5349,5350..5355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5370..5391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5508..5525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5679..6267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5759..5778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6438..7229,7230..7298))
                     /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     7061..7080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7299..7403)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7478..7497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccatg gtgagcaagg
      781 gcgaggggta ccatggtgag caagggggta ccatggtgag caagggcggg ggtaccaggt
      841 gagcaagggc gagggggtac catggtgagc aagggcgagg aggtcatcaa agagttcatg
      901 cgcttcaagg tgcgcatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      961 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc
     1021 cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac
     1081 gtgaagcacc ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag
     1141 tgggagcgcg tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc
     1201 ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac
     1261 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc
     1321 cgcgacggcg tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac
     1381 tacctggtgg agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac
     1441 tactacgtgg acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa
     1501 cagtacgagc gctccgaggg ccgccaccac ctgttcctgg ggcatggcac cggcagcacc
     1561 ggcagcggca gctccggcac cgcctcctcc gaggacaaca acatggccgt catcaaagag
     1621 ttcatgcgct tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag
     1681 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1741 ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag
     1801 gcgtacgtga agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc
     1861 ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac
     1921 tcctccctgc aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc
     1981 cccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg
     2041 tacccccgcg acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc
     2101 ggccactacc tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc
     2161 ggctactact acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc
     2221 gtggaacagt acgagcgctc cgagggccgc caccacctgt tcctgtacgg catggacgag
     2281 ctgtacaaga agcttacaag tttgtacaaa aagcaggctc caccatggga accaattcag
     2341 tcgacatgag ctggcttctg ttcctggccc acagagtcgc cttggccgcc ttgccctgcc
     2401 gccgcggctc tcgcgggttc gggatgttct atgccgtgag gaggggccgc aagaccgggg
     2461 tctttctgac ctggaatgag tgcagagcac aggtggaccg gtttcctgct gccagattta
     2521 agaagtttgc cacagaggat gaggcctggg cctttgtcag gaaatctgca agcccggaag
     2581 tttcagaagg gcatgaaaat caacatggac aagaatcgga ggcgaaagcc agcaagcgac
     2641 tccgtgagcc actggatgga gatggacatg aaagcgcaga gccgtatgca aagcacatga
     2701 agccgagcgt ggagccggcg cctccagtta gcagagacac gttttcctac atgggagact
     2761 tcgtcgtcgt ctacactgat ggctgctgct ccagtaatgg gcgtagaagg ccgcgagcag
     2821 gaatcggcgt ttactggggg ccgggccatc ctttaaatgt aggcattaga cttcctgggc
     2881 ggcagacaaa ccaaagagcg gaaattcatg cagcctgcaa agccattgaa caagcaaaga
     2941 ctcaaaacat caataaactg gttctgtata cagacagtat gtttacgata aatggtataa
     3001 ctaactgggt tcaaggttgg aagaaaaatg ggtggaagac aagtgcaggg aaagaggtga
     3061 tcaacaaaga ggactttgtg gcactggaga ggcttaccca ggggatggac attcagtgga
     3121 tgcatgttcc tggtcattcg ggatttatag gcaatgaaga agctgacaga ttagccagag
     3181 aaggagctaa acaatcggaa gactgagcgg ccgcactcga gatatctaga cccagctttc
     3241 ttgtacaaag tggttgatat ccagcacagt ggcggccgct caagtctaga gggcccgcgg
     3301 ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgct cgagtctaga
     3361 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3421 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3481 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3541 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3601 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     3661 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3721 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3781 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3841 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag
     3901 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag
     3961 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     4021 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     4081 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     4141 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
     4201 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     4261 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     4321 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     4381 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     4441 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     4501 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     4561 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     4621 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     4681 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     4741 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     4801 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     4861 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     4921 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     4981 gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5041 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5101 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5161 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5221 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5281 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5341 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5401 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5461 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5521 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5581 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5641 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5701 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5761 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5821 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5881 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5941 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6001 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6061 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6121 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6181 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6241 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6301 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6361 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     7441 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     7501 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     7561 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     7621 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
