LOCUS       sequence-403437-        8064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression Apobec1-ADAR2dd fusion protein as PIE-RBP control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8064)
  AUTHORS   Ruan X, Hu K, Zhang X
  TITLE     PIE-seq: identifying RNA-binding protein targets by dual 
            RNA-deaminase editing and sequencing.
  JOURNAL   Nat Commun. 2023 Jun 6;14(1):3275. doi: 10.1038/s41467-023-39054-8.
  PUBMED    37280234
REFERENCE   2  (bases 1 to 8064)
  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: "Nat 
            Commun."; date: "2023-06-6"; volume: "14(1):3275. doi"; pages: " 
            10.1038/s41467-023-39054-8"
COMMENT     Sequence Label: pR008_APAD
FEATURES             Location/Qualifiers
     source          1..8064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1735..1761
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1762..1785
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1795..2478
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     misc_feature    join(3715..4289,4290..4292,4293..4301)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3882..3899)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4109..4128
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4302..4304,4305..4307,4308..5021)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4302..4304,4305..4307,4308..5018)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4347..4368)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4608..4627)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4955..4976
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5085..5104)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5150..5205
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5204..5223)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5566..5582)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5566..5582)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5579..5601)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5590..5606
                     /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(5614..5620,5621..5638,5639..5644))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5659..5680
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5739..5934
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5785..5920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5940..6074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5977..5996)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6031..6050
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6142..6159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6313..6901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6393..6412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7072..7863,7864..7932))
                     /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     7695..7714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7933..8037)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcgcg ccaggcgggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt catgtaccca
     1741 tacgacgtcc cagactacgc tgactacaaa gacgatgacg acaagggcgc gcctagctca
     1801 gagactggcc cagtggctgt ggaccccaca ttgagacggc ggatcgagcc ccatgagttt
     1861 gaggtattct tcgatccgag agagctccgc aaggagacct gcctgcttta cgaaattaat
     1921 tgggggggcc ggcactccat ttggcgacat acatcacaga acactaacaa gcacgtcgaa
     1981 gtcaacttca tcgagaagtt cacgacagaa agatatttct gtccgaacac aaggtgcagc
     2041 attacctggt ttctcagctg gagcccatgc ggcgaatgta gtagggccat cactgaattc
     2101 ctgtcaaggt atccccacgt cactctgttt atttacatcg caaggctgta ccaccacgct
     2161 gacccccgca atcgacaagg cctgcgggat ttgatctctt caggtgtgac tatccaaatt
     2221 atgactgagc aggagtcagg atactgctgg agaaactttg tgaattatag cccgagtaat
     2281 gaagcccact ggcctaggta tccccatctg tgggtacgac tgtacgttct tgaactgtac
     2341 tgcatcatac tgggcctgcc tccttgtctc aacattctga gaaggaagca gccacagctg
     2401 acattcttta ccatcgctct tcagtcttgt cattaccagc gactgccccc acacattctc
     2461 tgggccaccg ggttgaaaag cggcagcgag actcccggga cctcagagtc cgccacaccc
     2521 gaaagtcagc tgcatttacc gcaggtttta gctgacgctg tctcacgcct ggtcctgggt
     2581 aagtttggtg acctgaccga caacttctcc tcccctcacg ctcgcagaaa agtgctggct
     2641 ggagtcgtca tgacaacagg cacagatgtt aaagatgcca aggtgataag tgtttctaca
     2701 ggaggcaaat gtattaatgg tgaatacatg agtgatcgtg gccttgcatt aaatgactgc
     2761 catgcagaaa taatatctcg gagatccttg ctcagatttc tttatacaca acttgagctt
     2821 tacttaaata acaaagatga tcaaaaaaga tccatctttc agaaatcaga gcgagggggg
     2881 tttaggctga aggagaatgt ccagtttcat ctgtacatca gcacctctcc ctgtggagat
     2941 gccagaatct tctcaccaca tgagccaatc ctggaagaac cagcagatag acacccaaat
     3001 cgtaaagcaa gaggacagct acggaccaaa atagagtctg gtcaggggac gattccagtg
     3061 cgctccaatg cgagcatcca aacgtgggac ggggtgctgc aaggggagcg gctgctcacc
     3121 atgtcctgca gtgacaagat tgcacgctgg aacgtggtgg gcatccaggg atcactgctc
     3181 agcattttcg tggagcccat ttacttctcg agcatcatcc tgggcagcct ttaccacggg
     3241 gaccaccttt ccagggccat gtaccagcgg atctccaaca tagaggacct gccacctctc
     3301 tacaccctca acaagccttt gctcagtggc atcagcaatg cagaagcacg gcagccaggg
     3361 aaggccccca acttcagtgt caactggacg gtaggcgact ccgctattga ggtcatcaac
     3421 gccacgactg ggaaggatga gctgggccgc gcgtcccgcc tgtgtaagca cgcgttgtac
     3481 tgtcgctgga tgcgtgtgca cggcaaggtt ccctcccact tactacgctc caagattacc
     3541 aagcccaacg tgtaccatga gtccaagctg gcggcaaagg agtaccaggc cgccaaggcg
     3601 cgtctgttca cagccttcat caaggcgggg ctgggggcct gggtggagaa gcccaccgag
     3661 caggaccagt tctcactcac gtaagcggcc gctacgtaaa ttccgccccc cccccccctc
     3721 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     3781 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     3841 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     3901 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     3961 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     4021 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     4081 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     4141 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     4201 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     4261 ggttttcctt tgaaaaacac gatgataata tggccacaac catggtgagc aagggcgagg
     4321 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4381 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4441 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4501 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4561 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4621 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4681 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4741 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4801 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4861 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     4921 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4981 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgg ccgccagcac
     5041 agtggtcgat cgaccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     5101 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     5161 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     5221 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     5281 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     5341 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     5401 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     5461 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     5521 ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     5581 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5641 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5701 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     5761 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5821 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     5881 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     5941 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     6001 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     6061 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6121 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6181 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6241 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6301 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6361 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6421 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6481 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6541 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6601 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6661 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6721 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6781 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6841 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6901 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6961 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7021 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7081 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7141 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7201 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7261 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7321 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7381 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7441 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7501 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7561 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7621 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7681 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7741 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7801 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7861 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7921 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7981 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     8041 atttccccga aaagtgccac ctgg
//
