LOCUS       sequence-494315-        8086 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Expresses rAPOBEC1-hADAR2dd-pAG in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8086)
  AUTHORS   Cheng QX, Xie G, Zhang X, Wang J, Ding S, Wu YX, Shi M, Duan FF, Wan
            ZL, Wei JJ, Xiao J, Wang Y
  TITLE     Co-profiling of in situ RNA-protein interactions and transcriptome 
            in single cells and tissues.
  JOURNAL   Nat Methods. 2025 Sep;22(9):1824-1835. doi: 
            10.1038/s41592-025-02774-4. Epub 2025 Aug 11.
  PUBMED    40784921
REFERENCE   2  (bases 1 to 8086)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-025-02774-4"; journalName: "Nat Methods"; date: 
            "2025-09"; volume: "22"; issue: "9"; pages: "1824-1835"
COMMENT     Sequence Label: pFastBac-rAPOBEC1-hADAR2dd-pAG
FEATURES             Location/Qualifiers
     source          1..8086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      102..556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     398..419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        582..686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(687..755,756..1547)
                     /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(905..924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1718..2306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2207..2226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2460..2477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2611..2835
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2902..3435)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3624..3629,3630..3646,3647..3652))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4160..4208
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4233..4252
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4344..4453)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4477..4568
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4493..4510
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             join(4620..4625,4626..4649,4650..4685,4686..4709)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4722..4742
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4752..5438
                     /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="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             6780..7205
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
     CDS             7221..7430
                     /codon_start=1
                     /product="immunoglobulin binding domain of Streptoccocus 
                     protein G"
                     /label=protein G
                     /translation="TTYKLVINGKTLKGETTTEAVDAETAERHFKQYANDNGVDGEWTY
                     DDATKTFTVTEKPEVIDASELTPAV"
     CDS             7455..7472
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7676..7810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7713..7732)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7767..7786
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7839..8004
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      361 ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
      421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2461 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2521 tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
     2581 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
     2641 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
     2701 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
     2761 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
     2821 gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
     2881 gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
     2941 ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
     3001 ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
     3061 cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
     3121 ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
     3181 ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
     3241 atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
     3301 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
     3361 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
     3421 gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
     3481 cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
     3541 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
     3601 agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc
     3661 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
     3721 aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
     3781 cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
     3841 aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
     3901 tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
     3961 atagttctag tggttggcta cgtacccgta gtggctatgg cagggcttgc cgccccgacg
     4021 ttggctgcga gccctgggcc ttcacccgaa cttgggggtt ggggtgggga aaaggaagaa
     4081 acgcgggcgt attggtccca atggggtctc ggtggggtat cgacagagtg ccagccctgg
     4141 gaccgaaccc cgcgtttatg aacaaacgac ccaacacccg tgcgttttat tctgtctttt
     4201 tattgccgtc atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct
     4261 cccccatctc ccggtaccgc atgctatgca tcagctgcta gcaccatggc tcgagatccc
     4321 gggtgatcaa gtcttcgtcg agtgattgta aataaaatgt aatttacagt atagtatttt
     4381 aattaatata caaatgattt gataataatt cttatttaac tataatatat tgtgttgggt
     4441 tgaattaaag gtccgtatac tccggaatat taatagatca tggagataat taaaatgata
     4501 accatctcgc aaataaataa gtattttact gttttcgtaa cagttttgta ataaaaaaac
     4561 ctataaatat tccggattat tcataccgtc ccaccatcgg gcgcggatct gccaccatgt
     4621 cagcttggtc tcaccctcag ttcgagaagg gcggtggatc tggaggtggt agcggaggct
     4681 ctgcctggag ccacccacag ttcgagaaga tcgatacgac cgaaaacctg tattttcagg
     4741 gatccatcac catgagctca gagactggcc cagtggctgt ggaccccaca ttgagacggc
     4801 ggatcgagcc ccatgagttt gaggtattct tcgatccgag agagctccgc aaggagacct
     4861 gcctgcttta cgaaattaat tgggggggcc ggcactccat ttggcgacat acatcacaga
     4921 acactaacaa gcacgtcgaa gtcaacttca tcgagaagtt cacgacagaa agatatttct
     4981 gtccgaacac aaggtgcagc attacctggt ttctcagctg gagcccatgc ggcgaatgta
     5041 gtagggccat cactgaattc ctgtcaaggt atccccacgt cactctgttt atttacatcg
     5101 caaggctgta ccaccacgct gacccccgca atcgacaagg cctgcgggat ttgatctctt
     5161 caggtgtgac tatccaaatt atgactgagc aggagtcagg atactgctgg agaaactttg
     5221 tgaattatag cccgagtaat gaagcccact ggcctaggta tccccatctg tgggtacgac
     5281 tgtacgttct tgaactgtac tgcatcatac tgggcctgcc tccttgtctc aacattctga
     5341 gaaggaagca gccacagctg acattcttta ccatcgctct tcagtcttgt cattaccagc
     5401 gactgccccc acacattctc tgggccaccg ggttgaaagc tagcagcggc agcgagactc
     5461 ccgggacctc agagtccgcc acacccgaaa gtcatatgtt gcacttggat cagacgccat
     5521 ctcgccagcc tattcccagt gagggtcttc agctgcattt accgcaggtt ttagctgacg
     5581 ctgtctcacg cctggtcctg ggtaagtttg gtgacctgac cgacaacttc tcctcccctc
     5641 acgctcgcag aaaagtgctg gctggagtcg tcatgacaac aggcacagat gttaaagatg
     5701 ccaaggtgat aagtgtttct acaggaacaa aatgtattaa tggtgaatac atgagtgatc
     5761 gtggccttgc attaaatgac tgccatgcag aaataatatc tcggagatcc ttgctcagat
     5821 ttctttatac acaacttgag ctttacttaa ataacaaaga tgatcaaaaa agatccatct
     5881 ttcagaaatc agagcgaggg gggtttaggc tgaaggagaa tgtccagttt catctgtaca
     5941 tcagcacctc tccctgtgga gatgccagaa tcttctcacc acatgagcca atcctggaag
     6001 aaccagcaga tagacaccca aatcgtaaag caagaggaca gctacggacc aaaatagagt
     6061 ctggtcaggg gacgattcca gtgcgctcca atgcgagcat ccaaacgtgg gacggggtgc
     6121 tgcaagggga gcggctgctc accatgtcct gcagtgacaa gattgcacgc tggaacgtgg
     6181 tgggcatcca gggatccctg ctcagcattt tcgtggagcc catttacttc tcgagcatca
     6241 tcctgggcag cctttaccac ggggaccacc tttccagggc catgtaccag cggatctcca
     6301 acatagagga cctgccacct ctctacaccc tcaacaagcc tttgctcagt ggcatcagca
     6361 atgcagaagc acggcagcca gggaaggccc ccaacttcag tgtcaactgg acggtaggcg
     6421 actccgctat tgaggtcatc aacgccacga ctgggaagga tgagctgggc cgcgcgtccc
     6481 gcctgtgtaa gcacgcgttg tactgtcgct ggatgcgtgt gcacggcaag gttccctccc
     6541 acttactacg ctccaagatt accaaaccca acgtgtacca tgagtccaag ctggcggcaa
     6601 aggagtacca ggccgccaag gcgcgtctgt tcacagcctt catcaaggcg gggctggggg
     6661 cctgggtgga gaagcccacc gagcaggacc agttctcact cacgcccagc ggcagcgaga
     6721 ctcccgggac ctcagagtcc gccacacccg aaagtggcgc gcccaccatg atcaccccct
     6781 ccctgaagga cgacccctcc cagtccgcca acctgctgtc cgaggccaag aagctgaacg
     6841 agtcccaggc ccccaaggcc gacaacaagt tcaacaagga gcagcagaac gccttctacg
     6901 agatcctgca cctgcccaac ctgaacgagg agcagcgcaa cggcttcatc cagtccctga
     6961 aggacgaccc tagccagagc gccaacctgc tggccgaggc caagaagctg aacgacgccc
     7021 aggcccccaa ggccgacaac aagttcaaca aggagcagca gaacgccttc tacgagatcc
     7081 tgcacctgcc taacctgacc gaggagcagc ggaacggctt catccagagc ctgaaggacg
     7141 accccagcgt gtccaaggag atcctggccg aggccaagaa gctgaacgac gcccaggccc
     7201 ctaagggcgg cggcggctcc accacctaca agctggtgat caacgggaag accctgaagg
     7261 gcgagaccac caccgaggcc gtggacgccg agaccgccga gagacacttc aagcagtacg
     7321 ccaacgacaa cggggtggac ggcgagtgga cctacgacga cgccaccaag accttcaccg
     7381 tgaccgagaa gcccgaggtg atcgacgcct ccgagctgac ccccgccgtg gacgacgaca
     7441 aggagttcgg tagccatcat caccatcacc attaagaatt caaaggccta cgtcgacgag
     7501 ctcactagtc gcggccgctt tcgaatctag agcctgcagt ctcgacaagc ttgtcgagaa
     7561 gtactagagg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     7621 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     7681 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     7741 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     7801 tgtctggatc tgatcactgc ttgagcctag gagatccgaa ccagataagt gaaatctagt
     7861 tccaaactat tttgtcattt ttaattttcg tattagctta cgacgctaca cccagttccc
     7921 atctattttg tcactcttcc ctaaataatc cttaaaaact ccatttccac ccctcccagt
     7981 tcccaactat tttgtccgcc cacagcgggg catttttctt cctgttatgt ttttaatcaa
     8041 acatcctgcc aactccatgt gacaaaccgt catcttcggc tacttt
//
