LOCUS       sequence-485483-        6522 bp    DNA     circular SYN 08-SEP-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 6522)
  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 6522)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 8, 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-HOXA9c (Npu)
FEATURES             Location/Qualifiers
     source          1..6522
                     /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             2062..2088
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2113..2130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2119..2343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2389..2817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2476..2495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2686..2707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2826..2846)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2831..3160
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3011..3146
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3073..3092
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3227..4021
                     /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(3281..3300)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3891..3910
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4195..4316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4232..4251)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4286..4305
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4365..4381)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4365..4381)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4378..4400)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4389..4405
                     /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(4413..4419,4420..4437,4438..4443))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4458..4479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4596..4613)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4767..5355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4847..4866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5526..6317,6318..6386))
                     /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     6149..6168
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6387..6491)
                     /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 agagaaaaac aacccagcga aggcgccttc tctgaaaaca atgctgagaa tgagagcggc
     1801 ggagacaagc cccccatcga tcccaataac ccagcagcca actggcttca tgcgcgctcc
     1861 actcggaaaa agcggtgccc ctatacaaaa caccagaccc tggaactgga gaaagagttt
     1921 ctgttcaaca tgtacctcac cagggaccgc aggtacgagg tggctcgact gctcaacctc
     1981 accgagaggc aggtcaagat ctggttccag aaccgcagga tgaaaatgaa gaaaatcaac
     2041 aaagaccgag caaaagacga gtacccgtac gacgttccgg actacgcttc ttgagtttaa
     2101 accgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2161 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2221 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2281 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2341 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2401 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2461 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2521 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2581 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2641 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2701 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2761 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2821 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2881 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2941 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3001 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3061 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3121 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3181 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3241 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3301 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3361 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3421 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3481 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3541 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3601 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3661 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3721 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3781 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3841 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3901 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3961 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4021 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4081 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4141 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4201 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4261 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4321 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4381 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4441 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4501 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4561 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4621 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4681 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4741 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4801 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4861 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4921 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4981 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5041 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5101 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5161 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5221 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5281 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5341 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5401 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5461 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5521 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5581 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5641 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5701 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5761 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5821 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5881 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5941 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6001 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6061 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6121 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6181 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6241 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6301 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6361 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6421 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6481 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
