LOCUS       sequence-324069-        5521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BEAR target cloning plasmid with split EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5521)
  AUTHORS   Talas A, Simon DA, Kulcsar PI, Varga E, Krausz SL, Welker E
  TITLE     BEAR reveals that increased fidelity variants can successfully 
            reduce the mismatch tolerance of adenine but not cytosine base 
            editors.
  JOURNAL   Nat Commun. 2021 Nov 3;12(1):6353. doi: 10.1038/s41467-021-26461-y.
  PUBMED    34732717
REFERENCE   2  (bases 1 to 5521)
  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: "2021-11-3"; volume: "12(1):6353. doi"; pages: " 
            10.1038/s41467-021-26461-y"
COMMENT     Sequence Label: pAT9624-BEAR-cloning
FEATURES             Location/Qualifiers
     source          1..5521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1709..1728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2056..2077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2120..2185
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2309..2430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2346..2365)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2400..2419
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2437..2892)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2574..2595)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2786..2805
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2919..3023
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3025..3382
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3025..3216
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3048..3068)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3233..3368
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3417..4211
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3417..4211
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3471..3490)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4081..4100
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4399..4418)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4443..4490
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4819..5407
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5308..5327
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtgcgagac
      901 gaggagatct tacgtctcaa ttttttagtt aaaatatggg aaagtttaaa agaaggaatg
      961 atgaataaac tatacaacca ctttaaatat gtgttggtcc cctttaagtc agcaagcaga
     1021 tatatcagat atggcagcta acttaaggag tccccgtgga tactggatga atagccccca
     1081 attccaccaa tgcaagaacc aacagtctat ttggtcttga ttaggacagt aaacttcctg
     1141 cacaccatag tctgtgcact aaacaggagt aaaacaccct taaggcatct cttacattgg
     1201 ctgcattaaa gtgtgtcact taatgacatc cgtgatatca gggatagttt gacattggac
     1261 atacagttct ctctcagtga ttgtcacctg cttatatagt acgaggaccc atatgtggtc
     1321 aatcactttc ttgttgccac tccatgccag ggaagacata gttacaatga gttataatta
     1381 taaaagctca gccttgcatg aggatataaa atacatttgc catacctcta catatgcctt
     1441 agttgctttt aagctttgtt tggaattgca aaggccaact tcctaaacac agtttcaaag
     1501 tattcttttg tcacaccttt ccactggata cttgtattgt ctgcaggaag aggccaccct
     1561 gcctatgctt ctccttaggg cctcgttctg cagagtcagc ataaactccc agtctgctca
     1621 accaattctg tctgattagg gccatatctc cttaccatga gttgattttt gtttcttttt
     1681 tgagcaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1741 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1801 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1861 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1921 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1981 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2041 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2101 catggacgag ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt
     2161 cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     2221 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2281 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2341 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2401 tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt
     2461 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2521 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2581 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2641 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2701 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2761 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2821 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2881 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3001 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3061 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3121 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3181 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3241 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3301 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3361 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3421 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3481 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3541 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3601 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3661 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3721 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3781 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3841 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3901 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3961 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4021 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4081 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4141 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4201 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4261 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4321 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4381 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4441 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4501 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     4561 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4621 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4681 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4741 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4801 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4861 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4921 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4981 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5041 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5101 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5161 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5221 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5281 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5341 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5401 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5461 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     5521 t
//
