LOCUS       sequence-494328-        8098 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Expresses rAPOBEC1-pAG-hADAR2dd in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8098)
  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 8098)
  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-pAG-hADAR2dd
FEATURES             Location/Qualifiers
     source          1..8098
                     /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             5514..5939
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
     CDS             5955..6164
                     /codon_start=1
                     /product="immunoglobulin binding domain of Streptoccocus 
                     protein G"
                     /label=protein G
                     /translation="TTYKLVINGKTLKGETTTEAVDAETAERHFKQYANDNGVDGEWTY
                     DDATKTFTVTEKPEVIDASELTPAV"
     CDS             7467..7484
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7688..7822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7725..7744)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7779..7798
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7851..8016
                     /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 gtcatatgac catgatcacc ccctccctga
     5521 aggacgaccc ctcccagtcc gccaacctgc tgtccgaggc caagaagctg aacgagtccc
     5581 aggcccccaa ggccgacaac aagttcaaca aggagcagca gaacgccttc tacgagatcc
     5641 tgcacctgcc caacctgaac gaggagcagc gcaacggctt catccagtcc ctgaaggacg
     5701 accctagcca gagcgccaac ctgctggccg aggccaagaa gctgaacgac gcccaggccc
     5761 ccaaggccga caacaagttc aacaaggagc agcagaacgc cttctacgag atcctgcacc
     5821 tgcctaacct gaccgaggag cagcggaacg gcttcatcca gagcctgaag gacgacccca
     5881 gcgtgtccaa ggagatcctg gccgaggcca agaagctgaa cgacgcccag gcccctaagg
     5941 gcggcggcgg ctccaccacc tacaagctgg tgatcaacgg gaagaccctg aagggcgaga
     6001 ccaccaccga ggccgtggac gccgagaccg ccgagagaca cttcaagcag tacgccaacg
     6061 acaacggggt ggacggcgag tggacctacg acgacgccac caagaccttc accgtgaccg
     6121 agaagcccga ggtgatcgac gcctccgagc tgacccccgc cgtggacgac gacaaggagt
     6181 tcactagtag cggcagcgag actcccggga cctcagagtc cgccacaccc gaaagtttgc
     6241 acttggatca gacgccatct cgccagccta ttcccagtga gggtcttcag ctgcatttac
     6301 cgcaggtttt agctgacgct gtctcacgcc tggtcctggg taagtttggt gacctgaccg
     6361 acaacttctc ctcccctcac gctcgcagaa aagtgctggc tggagtcgtc atgacaacag
     6421 gcacagatgt taaagatgcc aaggtgataa gtgtttctac aggaacaaaa tgtattaatg
     6481 gtgaatacat gagtgatcgt ggccttgcat taaatgactg ccatgcagaa ataatatctc
     6541 ggagatcctt gctcagattt ctttatacac aacttgagct ttacttaaat aacaaagatg
     6601 atcaaaaaag atccatcttt cagaaatcag agcgaggggg gtttaggctg aaggagaatg
     6661 tccagtttca tctgtacatc agcacctctc cctgtggaga tgccagaatc ttctcaccac
     6721 atgagccaat cctggaagaa ccagcagata gacacccaaa tcgtaaagca agaggacagc
     6781 tacggaccaa aatagagtct ggtcagggga cgattccagt gcgctccaat gcgagcatcc
     6841 aaacgtggga cggggtgctg caaggggagc ggctgctcac catgtcctgc agtgacaaga
     6901 ttgcacgctg gaacgtggtg ggcatccagg gatccctgct cagcattttc gtggagccca
     6961 tttacttctc gagcatcatc ctgggcagcc tttaccacgg ggaccacctt tccagggcca
     7021 tgtaccagcg gatctccaac atagaggacc tgccacctct ctacaccctc aacaagcctt
     7081 tgctcagtgg catcagcaat gcagaagcac ggcagccagg gaaggccccc aacttcagtg
     7141 tcaactggac ggtaggcgac tccgctattg aggtcatcaa cgccacgact gggaaggatg
     7201 agctgggccg cgcgtcccgc ctgtgtaagc acgcgttgta ctgtcgctgg atgcgtgtgc
     7261 acggcaaggt tccctcccac ttactacgct ccaagattac caaacccaac gtgtaccatg
     7321 agtccaagct ggcggcaaag gagtaccagg ccgccaaggc gcgtctgttc acagccttca
     7381 tcaaggcggg gctgggggcc tgggtggaga agcccaccga gcaggaccag ttctcactca
     7441 cgcccggcgg tagcggtagc aagcttcatc atcaccatca ccattaagaa ttcaaaggcc
     7501 tacgtcgacg agctcactag tcgcggccgc tttcgaatct agagcctgca gtctcgacaa
     7561 gcttgtcgag aagtactaga ggatcataat cagccatacc acatttgtag aggttttact
     7621 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     7681 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     7741 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     7801 tgtatcttat catgtctgga tctgatcact gcttgagcct aggagatccg aaccagataa
     7861 gtgaaatcta gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta
     7921 cacccagttc ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc
     7981 acccctccca gttcccaact attttgtccg cccacagcgg ggcatttttc ttcctgttat
     8041 gtttttaatc aaacatcctg ccaactccat gtgacaaacc gtcatcttcg gctacttt
//
