LOCUS       sequence-461147-        6746 bp    DNA     circular SYN 15-APR-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   Martinez DL, Todorovski I, Gonzalez MN, Rusimbi C, Blears D, Khallou
            N, Han Z, Dirac-Svejstrup B, Svejstrup JQ
  TITLE     bPAF1C allosterically activates CDK12/13 kinase during RNAPII 
            transcript elongation
  JOURNAL   bioRxiv 2024.10.14.618141
REFERENCE   2  (bases 1 to 6746)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.10.14.618141"
COMMENT     Sequence Label: pFRT-TO-FLAG(N)-CDC73
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(301..324)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(331..354)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(448..472)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    482..500
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    503..521
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(523..726)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(552..572)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        727..1106
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1278..1297
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1372..1476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1477..1545,1546..2337)
                     /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(1695..1714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2508..3096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2997..3016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3250..3267
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3384..3405
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3420..3425,3426..3443,3444..3450)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3458..3474
                     /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     3463..3485
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3482..3498
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3482..3498
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3547..3668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3558..3577)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3612..3631
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3798..4820)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(3798..4820)
                     /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(4828..4875)
                     /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     5007..5026
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5159..5383
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5372..5389
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 accattgaga tatccaagta gatcttttcg atcaggtctt ctaaccacag gaatattttc
       61 agtagctgca cgtcggacat aaacaggatg agaaaggtgc acgttattaa gtagaaataa
      121 aatggaatcc aatgtgtagt actctctggg ttggccttcc tttccagtcc cccaaacaac
      181 atagttggtc ttcacattct tgggccagga gaactccccg aagatcactt cgtctccctt
      241 caccacaatc tccttcttct ggatgttgta ctgtcgcagg acgctaagca cgtccgccat
      301 gggtccctga aagaggactt caagatcgag cttgtcatcg tcgtccttgt agtccataag
      361 cttaagttta aacgctagag tccggaggct ggatcggtcc cggtgtcttc tatggaggtc
      421 aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc actaaacgag ctcgtcgacg
      481 atctctatca ctgataggga gatctctatc actgataggg agagctctgc ttatatagac
      541 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
      601 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
      661 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
      721 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
      781 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
      841 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
      901 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
      961 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     1021 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     1081 ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag
     1141 cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc cttgccttgt
     1201 tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag
     1261 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc
     1321 gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     1381 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     1441 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     1501 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     1561 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     1621 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     1681 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     1741 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     1801 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     1861 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     1921 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     1981 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     2041 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     2101 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     2161 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     2221 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     2281 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     2341 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     2401 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     2461 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2521 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2581 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2641 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2701 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2761 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2821 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2881 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2941 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3001 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3061 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3121 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3181 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3241 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     3301 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3361 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     3421 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     3481 acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag
     3541 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3601 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3661 aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     3721 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     3781 aaatctcgta gtacgtgcta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca
     3841 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     3901 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     3961 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     4021 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     4081 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     4141 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     4201 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     4261 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     4321 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     4381 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     4441 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     4501 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     4561 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     4621 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     4681 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     4741 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     4801 gcggtgagtt caggcttttt ggccaaggaa gttcctatac tttctagaga ataggaactt
     4861 cggaatagga acttctaggt acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4921 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4981 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5041 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5101 ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc
     5161 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
     5221 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     5281 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     5341 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
     5401 gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatca tcagaacctt
     5461 aaatgggact tgtgttttac catgtacctg tccaatgttt cccaaaaccg taagaacact
     5521 ggtctatcca aatgacgttt gtgatagctg agttctaata ctgttacatc ccatttctga
     5581 acatttggat ccagacgaac ttcatcatac ttcagatgga aggctttaat tttagcaaat
     5641 atatcaactg gtgatccatc aggcaaaagc catggccaac ctttgaactg ccatgcagga
     5701 ccctgcacaa aaacggctac aacgcggtcc cagtcttgag gcataagttt aaggggctgg
     5761 tctactactc tataaggtac tgtaacacta attgcagtgc cccctggttg catctggtct
     5821 tttcttcttt gtattagagt ttcattttct cgttgacaac cttgtttctt cttttcatct
     5881 gatgggacaa atttcaggtc ctgtagaagg tcttttgcat taagcatggt tattaaagag
     5941 gtggtagctg caggaattat gataatgggt gttcgagatc ctttcttctg atttggggga
     6001 ggtcttgctt gagaaactgg tcttggtact ggctgggctg caggagtctg agtcttccgg
     6061 gcagatgcac cctccgttac agatttcagt gtcataccat ggtaggttcc catagtgtca
     6121 attttgaagc cttccgtttc ttcttttcct ttgaatcttt cctgatcgta tctgttatag
     6181 gcagctggga taggctgttt ggtgcgcaaa gtgggatcca caggtgctgc atttggggca
     6241 ggtcgctgtt caggtgcacg cccttcttct ctggctttta cagattgaag aattgcaaaa
     6301 atgttcttgg aaaaattctt tcctgtgctt tgtaagatag ttgttcgtgt cctccatact
     6361 ctctctctgc tgacaatatc tcgggtcaca tctacctcag catccacaaa actcctctgt
     6421 ttaagggcag ttatgtcatc atctagatca gtcttgatag tagatctttt cttagccata
     6481 attttggctt tgattgcagc aattttttcc actgacatag cttcagacaa agacctgatt
     6541 tgctccgttt gaacaatccc ttctttgtga ccctccaaac gggcagccaa tctctcttta
     6601 tcaaggcgca cacactcttc atcctcaatt cgtggtttct ttgcttctgc taaaacttca
     6661 tctgcagctc gtttgacttg agtagatcgc tgaagaccta tttctaaggg agcgcttctg
     6721 tctatacttg ccgatgttga cgcttc
//
