LOCUS       sequence-461141-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in Flp-IN human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  AUTHORS   Lopez Martinez D, Todorovski I, Noe Gonzalez M, Rusimbi C, Blears D,
            Khallou N, Han Z, Dirac-Svejstrup AB, Svejstrup JQ
  TITLE     PAF1C-mediated activation of CDK12/13 kinase activity is critical 
            for CTD phosphorylation and transcript elongation.
  JOURNAL   Mol Cell. 2025 May 15;85(10):1952-1967.e8. doi: 
            10.1016/j.molcel.2025.04.012. Epub 2025 May 1.
  PUBMED    40315851
REFERENCE   2  (bases 1 to 6746)
  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"; doi: 
            "10.1016/j.molcel.2025.04.012"; journalName: "Mol Cell"; date: 
            "2025-05-15- 15"; volume: "85"; issue: "10"; pages: "1952-1967.e8"
COMMENT     Sequence Label: pFRT-TO-FLAG(N)-CDC73-YY/AA
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(20..43)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(50..73)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(167..191)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    201..219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    222..240
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(242..445)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(271..291)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        446..825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     997..1016
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1091..1195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1196..1264,1265..2056)
                     /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(1414..1433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2227..2815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2716..2735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2969..2986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3103..3124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3139..3144,3145..3162,3163..3169)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3177..3193
                     /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     3182..3204
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3201..3217
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3201..3217
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3266..3387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3277..3296)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3331..3350
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3517..4539)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(3517..4539)
                     /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
                     "
     protein_bind    complement(4547..4594)
                     /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)."
     primer_bind     4726..4745
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4878..5102
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5091..5108
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 cgctaagcac gtccgccatg ggtccctgaa agaggacttc aagatcgagc ttgtcatcgt
       61 cgtccttgta gtccataagc ttaagtttaa acgctagagt ccggaggctg gatcggtccc
      121 ggtgtcttct atggaggtca aaacagcgtg gatggcgtct ccaggcgatc tgacggttca
      181 ctaaacgagc tcgtcgacga tctctatcac tgatagggag atctctatca ctgataggga
      241 gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
      301 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
      361 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
      421 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
      481 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
      541 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
      601 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
      661 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
      721 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
      781 accccgtaat tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg
      841 cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt
      901 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca
      961 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga
     1021 gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg
     1081 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1141 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1201 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1261 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1321 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1381 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1441 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1501 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1561 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1621 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1681 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     1741 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1801 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     1861 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     1921 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     1981 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2041 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2101 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2161 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2221 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2281 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2341 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2401 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2461 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2521 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2581 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2641 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2701 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2761 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2821 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2881 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2941 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3001 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3061 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3121 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3181 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct
     3241 agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact
     3301 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3361 accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc
     3421 gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata
     3481 gaaggcggcg gtggaatcga aatctcgtag tacgtgctat tcctttgccc tcggacgagt
     3541 gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc
     3601 cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc
     3661 gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata
     3721 gagttggtca agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc
     3781 cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca
     3841 gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat
     3901 gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac
     3961 gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc
     4021 gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc
     4081 aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg
     4141 gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac
     4201 cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc
     4261 acggcgggag atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg
     4321 aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc
     4381 ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg
     4441 agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag
     4501 aaacttctcg acagacgtcg cggtgagttc aggctttttg gccaaggaag ttcctatact
     4561 ttctagagaa taggaacttc ggaataggaa cttctaggta cgtgaaccat caccctaatc
     4621 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4681 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4741 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4801 cgccgcgctt aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt
     4861 tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     4921 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     4981 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     5041 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     5101 agtcgaggct gatcagcggg tttaaacggg ccctctagac tcgagcggcc gccactgtgc
     5161 tggatatcat cagaacctta aatgggactt gtgttttacc atgtacctgt ccaatgtttc
     5221 ccaaaaccgt aagaacactg gtctatccaa atgacgtttg tgatagctga gttctaatac
     5281 tgttacatcc catttctgaa catttggatc cagacgaact tcatcatact tcagatggaa
     5341 ggctttaatt ttagcaaata tatcaactgg tgatccatca ggcaaaagcc atggccaacc
     5401 tttgaactgc catgcaggac cctgcacaaa aacggctaca acgcggtccc agtcttgagg
     5461 cataagttta aggggctggt ctactactct ataaggtact gtaacactaa ttgcagtgcc
     5521 ccctggttgc atctggtctt ttcttctttg tattagagtt tcattttctc gttgacaacc
     5581 ttgtttcttc ttttcatctg atgggacaaa tttcaggtcc tgtagaaggt cttttgcatt
     5641 aagcatggtt attaaagagg tggtagctgc aggaattatg ataatgggtg ttcgagatcc
     5701 tttcttctga tttgggggag gtcttgcttg agaaactggt cttggtactg gctgggctgc
     5761 aggagtctga gtcttccggg cagatgcacc ctccgttaca gatttcagtg tcataccatg
     5821 gtaggttccc atagtgtcaa ttttgaagcc ttccgtttct tcttttcctt tgaatctttc
     5881 ctgatcggct ctgttagcgg cagctgggat aggctgtttg gtgcgcaaag tgggatccac
     5941 aggtgctgca tttggggcag gtcgctgttc aggtgcacgc ccttcttctc tggcttttac
     6001 agattgaaga attgcaaaaa tgttcttgga aaaattcttt cctgtgcttt gtaagatagt
     6061 tgttcgtgtc ctccatactc tctctctgct gacaatatct cgggtcacat ctacctcagc
     6121 atccacaaaa ctcctctgtt taagggcagt tatgtcatca tctagatcag tcttgatagt
     6181 agatcttttc ttagccataa ttttggcttt gattgcagca attttttcca ctgacatagc
     6241 ttcagacaaa gacctgattt gctccgtttg aacaatccct tctttgtgac cctccaaacg
     6301 ggcagccaat ctctctttat caaggcgcac acactcttca tcctcaattc gtggtttctt
     6361 tgcttctgct aaaacttcat ctgcagctcg tttgacttga gtagatcgct gaagacctat
     6421 ttctaaggga gcgcttctgt ctatacttgc cgatgttgac gcttcaccat tgagatatcc
     6481 aagtagatct tttcgatcag gtcttctaac cacaggaata ttttcagtag ctgcacgtcg
     6541 gacataaaca ggatgagaaa ggtgcacgtt attaagtaga aataaaatgg aatccaatgt
     6601 gtagtactct ctgggttggc cttcctttcc agtcccccaa acaacatagt tggtcttcac
     6661 attcttgggc caggagaact ccccgaagat cacttcgtct cccttcacca caatctcctt
     6721 cttctggatg ttgtactgtc gcagga
//
