LOCUS       sequence-394801-        7386 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of domain inlaid Nme2-ABE8e-i7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7386)
  AUTHORS   Bamidele N, Zhang H, Dong X, Cheng H, Gaston N, Feinzig H, Cao H, 
            Kelly K, Watts JK, Xie J, Gao G, Sontheimer EJ
  TITLE     Domain-inlaid Nme2Cas9 adenine base editors with improved activity 
            and targeting scope.
  JOURNAL   Nat Commun. 2024 Feb 17;15(1):1458. doi: 10.1038/s41467-024-45763-5.
  PUBMED    38368418
REFERENCE   2  (bases 1 to 7386)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-02-17"; volume: "15(1):1458. doi"; pages: " 
            10.1038/s41467-024-45763-5"
COMMENT     Sequence Label: pEJS2164_Nme2-ABE8e-i7
FEATURES             Location/Qualifiers
     source          1..7386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     610..629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        610..628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             678..698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4605..4625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4634..4651
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4674..4691)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4680..4904
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4975..4991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4975..4991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4988..5010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4999..5015
                     /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(5023..5029,5030..5047,5048..5053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5068..5089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5206..5223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5377..5965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5457..5476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6136..6927,6928..6996))
                     /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     6759..6778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6997..7101)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7180..7199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7371..7386,1..364)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
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 gctagagatc
      601 cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg acagccgacg
      661 gaagcgagtt cgagtcacca aagaagaagc ggaaagtcga agatatggcc gccttcaagc
      721 ctaacccaat caattacatc ctgggactgg ccatcggaat cgcatccgtg ggatgggcta
      781 tggtggagat cgacgaggag gagaatccta tccggctgat cgatctgggc gtgagagtgt
      841 ttgagagggc cgaggtgcca aagaccggcg attctctggc tatggcccgg agactggcac
      901 ggagcgtgag gcgcctgaca cggagaaggg cacacaggct gctgagggca cgccggctgc
      961 tgaagagaga gggcgtgctg caggcagcag acttcgatga gaatggcctg atcaagagcc
     1021 tgccaaacac cccctggcag ctgagagcag ccgccctgga caggaagctg acaccactgg
     1081 agtggtctgc cgtgctgctg cacctgatca agcaccgcgg ctacctgagc cagcggaaga
     1141 acgagggaga gacagcagac aaggagctgg gcgccctgct gaagggagtg gccaacaatg
     1201 cccacgccct gcagaccggc gatttcagga cacctgccga gctggccctg aataagtttg
     1261 agaaggagtc cggccacatc agaaaccaga ggggcgacta tagccacacc ttctcccgca
     1321 aggatctgca ggccgagctg atcctgctgt tcgagaagca gaaggagttt ggcaatccac
     1381 acgtgagcgg aggcctgaag gagggaatcg agaccctgct gatgacacag aggcctgccc
     1441 tgtccggcga cgcagtgcag aagatgctgg gacactgcac cttcgagcct gcagagccaa
     1501 aggccgccaa gaacacctac acagccgagc ggtttatctg gctgacaaag ctgaacaatc
     1561 tgagaatcct ggagcaggga tccgagaggc cactgaccga cacagagagg gccaccctga
     1621 tggatgagcc ttaccggaag tctaagctga catatgccca ggccagaaag ctgctgggcc
     1681 tggaggacac cgccttcttt aagggcctga gatacggcaa ggataatgcc gaggcctcca
     1741 cactgatgga gatgaaggcc tatcacgcca tctctcgcgc cctggagaag gagggcctga
     1801 aggacaagaa gtcccccctg aacctgagct ccgagctgca ggatgagatc ggcaccgcct
     1861 tctctctgtt taagaccgac gaggatatca caggccgcct gaaggacagg gtgcagcctg
     1921 agatcctgga ggccctgctg aagcacatct ctttcgataa gtttgtgcag atcagcctga
     1981 aggccctgag aaggatcgtg ccactgatgg agcagggcaa gcggtacgac gaggcctgcg
     2041 ccgagatcta cggcgatcac tatggcaaga agaacacaga ggagaagatc tatctgcccc
     2101 ctatccctgc cgacgagatc agaaatcctg tggtgctgag ggccctgtcc caggcaagaa
     2161 aagtgatcaa cggagtggtg cgccggtacg gatctccagc ccggatccac atcgagaccg
     2221 ccagagaagt gggcaagagc ttcaaggacc ggaaggagat cgagaagaga caggaggaga
     2281 atcgcaagga tcgggagaag gccgccgcca agtttaggga gtacttccct aactttgtgg
     2341 gcgagccaaa gtctaaggac atcctgaagc tgcgcctgta cgagcagcag cacggcaagt
     2401 gtctgtatag cggcaaggag atcaatctgg tgcggctgaa cgagaagggc tatgtggaga
     2461 tcgatcacgc cctgcctttc tccagaacct gggacgattc ttttaacaat aaggtgctgg
     2521 tgctgggcag cgagaaccag aataagggca atcagacacc atacgagtat ttcaatggca
     2581 aggacaactc cagggagtgg caggagttca aggcccgcgt ggagacctct agatttccca
     2641 ggagcaagaa gcagcggatc ctgctgcaga agttcgacga ggatggcttt aaggagtgca
     2701 acctgaatga caccagatac gtgaaccggt tcctgtgcca gtttgtggcc gatcacatcc
     2761 tgctgaccgg caagggcaag agaagggtgt tcgcctctaa tggccagatc acaaacctgc
     2821 tgaggggatt ttggggactg aggaaggtgc gggcagagaa tgacagacac cacgcactgg
     2881 atgcagtggt ggtggcatgc agcaccgtgg caatgcagca gaagatcaca agattcgtga
     2941 ggtataagga gatgaacgcc tttgacggca agaccatcga taaggagggc ggatcaggag
     3001 gctctggcgg ttcaggtgga tcaggcggta gcggaggttc aggtggttct gaggtggagt
     3061 tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca cgcgatgaga
     3121 gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc gagggctgga
     3181 acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc ctgagacagg
     3241 gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg acattcgagc
     3301 cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg gtgtttggcg
     3361 tgaggaacag caaacggggc gccgcaggct ccctgatgaa cgtgctgaac taccccggca
     3421 tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc gccctgctgt
     3481 gcgacttcta ccggatgcct agacaggtgt tcaatgctca gaagaaggcc cagagctcca
     3541 tcaacggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca cctgaaagca
     3601 gcggcacagg caaggtgctg caccagaaga cccacttccc ccagccttgg gagttctttg
     3661 cccaggaagt gatgatccgg gtgttcggca agccagacgg caagcctgag tttgaggagg
     3721 ccgatacccc agagaagctg aggacactgc tggcagagaa gctgtctagc aggccagagg
     3781 cagtgcacga gtacgtgacc ccactgttcg tgtccagggc acccaatcgg aagatgtctg
     3841 gcgcccacaa ggacacactg agaagcgcca agaggtttgt gaagcacaac gagaagatct
     3901 ccgtgaagag agtgtggctg accgagatca agctggccga tctggagaac atggtgaatt
     3961 acaagaacgg cagggagatc gagctgtatg aggccctgaa ggcaaggctg gaggcctacg
     4021 gaggaaatgc caagcaggcc ttcgacccaa aggataaccc cttttataag aagggaggac
     4081 agctggtgaa ggccgtgcgg gtggagaaga cccaggagag cggcgtgctg ctgaataaga
     4141 agaacgccta cacaatcgcc gacaatggcg atatggtgag agtggacgtg ttctgtaagg
     4201 tggataagaa gggcaagaat cagtacttta tcgtgcctat ctatgcctgg caggtggccg
     4261 agaacatcct gccagacatc gattgcaagg gctacagaat cgacgatagc tatacattct
     4321 gtttttccct gcacaagtat gacctgatcg ccttccagaa ggatgagaag tccaaggtgg
     4381 agtttgccta ctatatcaat tgcgactcct ctaacggcag gttctacctg gcctggcacg
     4441 ataagggcag caaggagcag cagtttcgca tctccaccca gaatctggtg ctgatccaga
     4501 agtatcaggt gaacgagctg ggcaaggaga tcaggccatg tcggctgaag aagcgcccac
     4561 ccgtgcggga ggataaaaga accgccgacg gcagcgaatt cgagcccaag aagaagagga
     4621 aagtctaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
     4681 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4741 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4801 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4861 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     4921 aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4981 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5041 cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5101 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5161 acgcgcgggg agaggcggtt tgcgtattgg gcgcacttcc gcttcctcgc tcactgactc
     5221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacac
     6001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6241 cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga
     6301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     7141 gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct ctgatgccgc
     7201 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     7261 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     7321 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     7381 attgac
//
