LOCUS       sequence-416783-        5808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-tagged Drosophila mts protein with D85N mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5808)
  AUTHORS   Fujiwara R, Zhai SN, Liang D, Shah AP, Tracey M, Ma XK, Fields CJ, 
            Mendoza-Figueroa MS, Meline MC, Tatomer DC, Yang L, Wilusz JE
  TITLE     IntS6 and the Integrator phosphatase module tune the efficiency of 
            select premature transcription termination events.
  JOURNAL   Mol Cell. 2023 Nov 14:S1097-2765(23)00902-4. doi: 
            10.1016/j.molcel.2023.10.035.
  PUBMED    37995689
REFERENCE   2  (bases 1 to 5808)
  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: "Mol Cell. 
            2023 Nov 14:S1097-2765(23)00902-4. doi: 
            10.1016/j.molcel.2023.10.035."
COMMENT     Sequence Label: pUb FLAG-Mts D85N
FEATURES             Location/Qualifiers
     source          1..5808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     147..166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     400..417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    534..555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(570..575,576..593,594..600)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    608..624
                     /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)."
     primer_bind     613..635
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     632..648
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     632..648
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2709..2774
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3736..3777
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3787..3804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3822..3951
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     primer_bind     complement(4065..4084)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4184..4206
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4244..4262)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4330..4434
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4435..4503,4504..5295)
                     /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     complement(4653..4672)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5466..5808,1..246)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
       61 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
      421 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      481 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      541 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      601 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      661 aagctttgtc gccggaacgc agcgacagag attccaatgt gtccgtatct ttcaggcttt
      721 tgcccttcag ttccagacga agcgactggc gattcgcgtg tggggtctgc ttcagggtct
      781 tgtgaattag ggcgcgcaga tcgccgatgg gcgtggcgcc ggagggcacc ttcaccttgc
      841 cgtacggctt gctgttcttc gcgttcaaaa tctccagctc cattttgctt tcggtgcgct
      901 tgcaatcagt actgtccaaa atcgaaaatc gccgaaccgt agtgtgaccg tgcggggctc
      961 tgcgaaaata aactttttta ggtatatggc cacacacggg gaaagcacag tggattatat
     1021 gttttaatat tataatatgc aggttttcat tacttatcca gatgtaagcc cacttaaagc
     1081 gatttaacaa ttatttgccg aaagagtata aacaaatttc acttaaaaat ggattaagaa
     1141 aagctagctt gtgtaagatt atgcgcagcg ttgccagata gctccattta aaacacttca
     1201 aaaacaataa gttttgaaaa tatatacata aatagcagtc gttgccgcaa cgctcaacac
     1261 atcacacttt taaaacaccc tttacctaca cagaattact ttttaaattt ccagtcaagc
     1321 tgcgagtttc aaaattatag ccggtagaga agacagtgct atttcaaaag caaactaaca
     1381 agggtcttaa attccaaaac accaatccta acaagccttg gacttttgta agtttagatc
     1441 aaaggtggca ttgcattcaa tgtcatggta agaagtaggt cgtctaggta gaaatcctca
     1501 ttcagccggt caagtcagta cgagaaaggt ctcaatttga aattgtctta aaaatatttt
     1561 attgttttgt actgtggtga gtttaaacga aaaacacaaa aaaaaagtga tacacagaaa
     1621 tcataaaaaa ttttaataca aggtattcgt acgtatcaaa aacatttcgg cacaattttt
     1681 tttctctgta ctaaagtgtt acgaacacta cggtattttt tagtgatttt caacggacac
     1741 cgaaggtata taaacagcgt tcgcgaacgg tcgccttcaa aaccaattga catttgcagc
     1801 agcaagtaca agtagaaagt aaagcgcaat cagcgaaaaa tttatactta attgttggtg
     1861 attaaagtac aattaaaaga acattctcga aagtcacaag aaacgtaagt ttttaactcg
     1921 ctgttaccaa ttagtaataa gagcaacaag acgttgagta atttcaagaa aaactgcatt
     1981 tcaaggtctt tgttcggcca ttttttttta ttcaacgctc tacgtaatta caaaataaga
     2041 aattggcagc cacgcatctt gttttcccaa tgaattggca tcaaaacgca aacaaatcta
     2101 taaataaaac ttgcgtgttg attttcgcca agatttattg gcaaattgtg aaattcgcag
     2161 tgacgcattt gaaaattcga gaaatcacga acgcactcga tcgagcattt gtgtgcatgt
     2221 tattagttag ttgtttagtt aattgaagta ttttaccaac gaaatccact tatttttagc
     2281 tgaaatagag taggttgctt aaacaaagcc acgtctgaaa atttcttatt gcttgtagtt
     2341 gtgacgtcac catatacaca caaaataatg tgtatgcatg catttcagct gtgtatatat
     2401 acatgcacac actcgcaaca cgaaaacgat gacgaagcaa cggaacaaag gtttctcaac
     2461 taccctttgt tccctgtttc ttcgctttcc tttgttccaa tattcgtaga gggttaatag
     2521 gggtttctca acaaagttgg cgtcgataaa taagtttccc atttttattc cccagccagg
     2581 aagttagttt caatagtttt gtaatttcaa cgaaactcat ttgatttcgt actaattttc
     2641 cacatctcta ttttctgccc gcagaataat ccaaactgca ggtcgactct agctagagga
     2701 agcttatgga ctacaaagac catgacggtg attataaaga tcatgatatc gattacaagg
     2761 atgacgatga caaggatcca ctagtcgagg ataaagcaac aacaaaagat cttgatcaat
     2821 ggattgagca gttgaacgaa tgcaatcagt tgacagagac acaagttcgc accctctgcg
     2881 acaaggccaa ggagattctc tccaaggagt ccaatgtgca ggaggtaaaa tgcccggtga
     2941 cagtgtgcgg agatgtccac ggtcagtttc acgacctcat ggagctcttc cggataggcg
     3001 gcaagtctcc ggacaccaac tacctgttca tgggcaacta cgtggaccgt ggatactact
     3061 ccgtggagac cgtgaccctt ctggtggccc tgaaggttcg ctatcgcgag cgcatcacca
     3121 tcctgcgcgg taaccacgag tcgcgccaga tcacacaggt gtacggcttc tacgacgagt
     3181 gcctgcgcaa gtatggcaat gccaacgttt ggaagtactt cacggatctg ttcgactact
     3241 tgccactgac ggcactcgtc gacgggcaga tcttctgcct gcacggaggc ctcagtccct
     3301 cgatcgacag tctggatcac attcgggccc tcgatcgctt gcaggaggtt ccgcacgagg
     3361 gtcccatgtg cgatctgctc tggtccgatc ccgatgacag gggtggctgg ggaatctcgc
     3421 ctcgtggcgc cggttacacc tttggccagg atatttcgga aacctttaac aacacaaacg
     3481 gcctgacact ggtgtcgcgc gcccatcagc tggtgatgga gggctacaac tggtgtcacg
     3541 atcgcaatgt ggtcacaata ttctcagcgc caaactattg ctaccgctgt ggcaaccaag
     3601 cggctcttat ggaactggat gattcactta aattttcatt cctacaattt gacccagcac
     3661 ccaggcgcgg ggagcctcat gttacgcgaa gaacacccga ttatttcctt taatctagag
     3721 ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt
     3781 accggtcatc atcaccatca ccattgagtt tatctgacta aatcttagtt tgtattgtca
     3841 tgttttaata caatatgtta tgtttaaata tgtttttaat aaattttata aaataatttc
     3901 aacttttatt gtaacaacat tgtccattta cacactcctt tcaagcgcgt gggatcgatg
     3961 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4021 tgtgagccat atgggcccat gtgagccata tggtgcactc tcagtacaat ctgctctgat
     4081 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4141 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4201 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4261 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4321 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4381 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4441 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4501 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4561 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4621 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4681 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4741 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4801 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4861 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4921 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4981 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5041 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5101 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5161 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5221 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5281 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5341 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5401 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5461 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5521 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5581 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5641 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5701 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5761 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagccc
//
