LOCUS       sequence-485490-        6471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Proximity-triggered Protein Trans-Splicing to generate the splice 
            product MLL-AF9, Extended Figure 4 in Nature Chemical Biology 20.10 
            (2024): 1353-1360..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  AUTHORS   Lee G, Muir TW
  TITLE     Distinct phases of cellular signaling revealed by time-resolved 
            protein synthesis.
  JOURNAL   bioRxiv [Preprint]. 2023 Jul 10:2023.07.10.548208. doi: 
            10.1101/2023.07.10.548208.
  PUBMED    37503273
REFERENCE   2  (bases 1 to 6471)
  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: "bioRxiv 
            [Preprint]. 2023 Jul 10:2023.07.10.548208. doi: 
            10.1101/2023.07.10.548208."
COMMENT     Sequence Label: pcDNA3.1-NES-CAGEc-AF9c (Npu)
FEATURES             Location/Qualifiers
     source          1..6471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             988..1011
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1021..1044
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1054..1077
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1114..1389
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRIS"
     CDS             1573..1593
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1594..1614
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2011..2037
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2062..2079)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2068..2292
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2338..2766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2425..2444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2635..2656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2775..2795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2780..3109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2960..3095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3022..3041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3176..3970
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3230..3249)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3840..3859
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4144..4265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4181..4200)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4235..4254
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4314..4330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4314..4330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4327..4349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4338..4354
                     /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(4362..4368,4369..4386,4387..4392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4407..4428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4545..4562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4716..5304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4796..4815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5475..6266,6267..6335))
                     /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     6098..6117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6336..6440)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccatggctc tccaaaaaaa gttggaagag cttgaactgg acgaacaggg aggaagtgga
      961 ggtagtctcg agggtaccat gggttcagac tataaagatg atgatgataa aggtggctcc
     1021 gactacaagg atgacgatga caagggaggt agtgactaca aagacgatga cgacaagggc
     1081 ggaggaggaa gtggaggtag tggaagagta gcgatcctct ggcatgagat gtggcatgaa
     1141 ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg catgtttgag
     1201 gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa ggaaacatcc
     1261 tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag gaagtacatg
     1321 aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca tgtgttccga
     1381 cgaatctcag gaaatggaaa tggaggagaa caagaggtgt ttgagtattg tttggaagat
     1441 ggttcattga ttcgggcaac aaaagaccat aagtttatga ctgttgatgg tcaaatgttg
     1501 ccaattgatg aaatatttga acgtgaattg gatttgatgc gggttgataa tttgccgaat
     1561 ggaagtggag gagagaacct gtattttcag ggcgagaacc tgtattttca gggcggaagt
     1621 ggaggaatca aaatagccac acgtaaatat ttaggcaaac aaaatgtcta tgacattgga
     1681 gttgagcgcg accataattt tgcactcaaa aatggcttca tagcttctaa ttgctttaat
     1741 cttgaagtga aaagtccaat aaagcaaagc aaatcagata agcaaataaa gaatggtgaa
     1801 tgtgacaagg catacctaga tgaactggta gagcttcaca gaaggttaat gacattgaga
     1861 gaaagacaca ttctgcagca gatcgtgaac cttatagaag aaactggaca ctttcatatc
     1921 acaaacacaa catttgattt tgatctttgc tcgctggaca aaaccacagt ccgtaaacta
     1981 cagagttacc tggaaacatc tggaacatcc tacccgtacg acgttccaga ctacgcttct
     2041 tgagtttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2101 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2161 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2221 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2281 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2341 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2401 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2461 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2521 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2581 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2641 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2701 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2761 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2821 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2881 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     2941 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3001 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3061 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3121 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3181 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3241 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3301 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3361 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3421 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3481 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3541 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3601 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3661 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     3721 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     3781 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     3841 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3901 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     3961 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4021 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4081 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4141 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4201 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4261 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4321 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4381 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4501 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4561 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4621 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4681 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4741 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4801 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4861 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4921 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4981 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5041 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5101 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5161 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5221 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5281 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5341 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5401 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5461 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5521 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5581 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5641 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5701 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5761 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5821 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5881 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5941 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6001 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6061 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6121 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6181 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6241 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6301 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6361 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6421 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
