LOCUS       Exported                9273 bp ds-DNA     circular SYN 27-SEP-2021
DEFINITION  Mammalian expression of SaCas9 prime editor 2 with P2A-BSD marker.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-SaPE2-P2A-BSD
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9273)
  TITLE     Enhanced Prime Editing Systems Through Identification and 
            Manipulation of Cellular Determinants of Editing Outcomes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9273)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 27, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9273
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        135..514
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..718
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     669..689
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     760..779
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        760..778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             828..848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6180..6200
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6210..6266
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6267..6665
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             6671..6688
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6711..6728)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6717..6941
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7012..7028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7012..7028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7025..7047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7036..7052
                     /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(7060..7090)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7105..7126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7243..7260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7414..8002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7494..7513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8173..9033)
                     /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     8796..8815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9034..9138)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9217..9236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
       61 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      121 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      181 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      241 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      301 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      361 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      421 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      481 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      541 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      601 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      661 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      721 tttagtgaac cgtcagatcc gctagagatc cgcggccgct aatacgactc actataggga
      781 gagccgccac catgaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc
      841 ggaaagtcgg gaagcgaaat tacattctgg ggctggacat tggcattaca tcagtgggct
      901 atggcatcat tgactacgag acaagggacg tgatcgacgc cggcgtgaga ctgttcaagg
      961 aggccaacgt ggagaacaat gagggccgga gatccaagag gggagcaagg cgcctgaagc
     1021 ggagaaggcg ccacagaatc cagagagtga agaagctgct gttcgattac aacctgctga
     1081 ccgaccactc cgagctgtct ggcatcaatc cttatgaggc cagagtgaag ggcctgtccc
     1141 agaagctgtc tgaggaggag tttagcgccg ccctgctgca cctggcaaag aggagaggcg
     1201 tgcacaacgt gaatgaggtg gaggaggaca ccggcaacga gctgtccaca aaggagcaga
     1261 tcagccgcaa ttccaaggcc ctggaggaga agtatgtggc cgagctgcag ctggagcggc
     1321 tgaagaagga tggcgaggtg aggggctcca tcaatcgctt caagacctct gactacgtga
     1381 aggaggccaa gcagctgctg aaggtgcaga aggcctacca ccagctggat cagtccttta
     1441 tcgatacata tatcgacctg ctggagacaa ggcgcacata ctatgaggga ccaggagagg
     1501 gctctccctt cggctggaag gacatcaagg agtggtacga gatgctgatg ggccactgca
     1561 cctattttcc agaggagctg agaagcgtga agtacgccta taacgccgat ctgtacaacg
     1621 ccctgaatga cctgaacaac ctggtcatca ccagggatga gaacgagaag ctggagtact
     1681 atgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagcct acactgaagc
     1741 agatcgccaa ggagatcctg gtgaacgagg aggacatcaa gggctaccgc gtgacctcca
     1801 caggcaagcc agagttcacc aatctgaagg tgtatcacga tatcaaggac atcacagccc
     1861 ggaaggagat catcgagaac gccgagctgc tggatcagat cgccaagatc ctgaccatct
     1921 atcagagctc cgaggacatc caggaggagc tgaccaacct gaatagcgag ctgacacagg
     1981 aggagatcga gcagatcagc aatctgaagg gctacaccgg cacacacaac ctgagcctga
     2041 aggccatcaa tctgatcctg gatgagctgt ggcacacaaa cgacaatcag atcgccatct
     2101 ttaaccggct gaagctggtg ccaaagaagg tggacctgtc ccagcagaag gagatcccaa
     2161 ccacactggt ggacgatttc atcctgtctc ccgtggtgaa gcggagcttc atccagagca
     2221 tcaaagtgat caacgccatc atcaagaagt acggcctgcc caatgatatc atcatcgagc
     2281 tggccaggga gaagaactcc aaggacgccc agaagatgat caatgagatg cagaagagga
     2341 accgccagac caatgagcgg atcgaggaga tcatcagaac cacaggcaag gagaacgcca
     2401 agtacctgat cgagaagatc aagctgcacg atatgcagga gggcaagtgt ctgtattctc
     2461 tggaggccat ccctctggag gacctgctga acaatccatt caactacgag gtggatcaca
     2521 tcatcccccg gagcgtgagc ttcgacaatt cttttaacaa taaggtgctg gtgaagcagg
     2581 aggaggccag caagaagggc aataggaccc ctttccagta cctgtctagc tccgattcta
     2641 agatcagcta cgagacattc aagaagcaca tcctgaatct ggccaagggc aagggccgca
     2701 tcagcaagac caagaaggag tacctgctgg aggagcggga catcaacaga ttctccgtgc
     2761 agaaggactt catcaaccgg aatctggtgg acaccagata cgccacacgc ggcctgatga
     2821 atctgctgcg gtcttatttc agagtgaaca atctggatgt gaaggtgaag agcatcaacg
     2881 gcggcttcac ctcctttctg cggagaaagt ggaagtttaa gaaggagcgc aacaagggct
     2941 ataagcacca cgccgaggat gccctgatca tcgccaatgc cgacttcatc tttaaggagt
     3001 ggaagaagct ggacaaggcc aagaaagtga tggagaacca gatgttcgag gagaagcagg
     3061 ccgagagcat gcccgagatc gagacagagc aggagtacaa ggagattttc atcacacctc
     3121 accagatcaa gcacatcaag gacttcaagg actacaagta ttctcacagg gtggataaga
     3181 agcccaaccg cgagctgatc aatgacaccc tgtatagcac acggaaggac gataagggca
     3241 ataccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggataatgac aagctgaaga
     3301 agctgatcaa caagtctccc gagaagctgc tgatgtacca ccacgatcct cagacatatc
     3361 agaagctgaa gctgatcatg gagcagtacg gcgacgagaa gaacccactg tataagtact
     3421 atgaggagac aggcaactac ctgacaaagt atagcaagaa ggataatggc cccgtgatca
     3481 agaagatcaa gtactatggc aacaagctga atgcccacct ggacatcacc gacgattacc
     3541 ctaactctcg caataaggtg gtgaagctga gcctgaagcc ataccggttc gacgtgtacc
     3601 tggacaacgg cgtgtataag tttgtgacag tgaagaatct ggatgtgatc aagaaggaga
     3661 actactatga ggtgaacagc aagtgctacg aggaggccaa gaagctgaag aagatcagca
     3721 accaggccga gttcatcgcc tctttttaca acaatgacct gatcaagatc aatggcgagc
     3781 tgtatagagt gatcggcgtg aacaatgatc tgctgaacag aatcgaagtg aatatgatcg
     3841 acatcaccta cagggagtat ctggagaaca tgaatgataa gaggccccct cgcatcatca
     3901 agaccatcgc ctctaagaca cagagcatca agaagtacag cacagacatc ctggggaacc
     3961 tgtatgaagt caagagcaag aaacatcctc agattatcaa gaaaggctct ggaggatcta
     4021 gcggaggatc ctctggcagc gagacaccag gaacaagcga gtcagcaaca ccagagagca
     4081 gtggcggcag cagcggcggc agcagcaccc taaatataga agatgagtat cggctacatg
     4141 agacctcaaa agagccagat gtttctctag ggtccacatg gctgtctgat tttcctcagg
     4201 cctgggcgga aaccgggggc atgggactgg cagttcgcca agctcctctg atcatacctc
     4261 tgaaagcaac ctctaccccc gtgtccataa aacaataccc catgtcacaa gaagccagac
     4321 tggggatcaa gccccacata cagagactgt tggaccaggg aatactggta ccctgccagt
     4381 ccccctggaa cacgcccctg ctacccgtta agaaaccagg gactaatgat tataggcctg
     4441 tccaggatct gagagaagtc aacaagcggg tggaagacat ccaccccacc gtgcccaacc
     4501 cttacaacct cttgagcggg ctcccaccgt cccaccagtg gtacactgtg cttgatttaa
     4561 aggatgcctt tttctgcctg agactccacc ccaccagtca gcctctcttc gcctttgagt
     4621 ggagagatcc agagatggga atctcaggac aattgacctg gaccagactc ccacagggtt
     4681 tcaaaaacag tcccaccctg tttaatgagg cactgcacag agacctagca gacttccgga
     4741 tccagcaccc agacttgatc ctgctacagt acgtggatga cttactgctg gccgccactt
     4801 ctgagctaga ctgccaacaa ggtactcggg ccctgttaca aaccctaggg aacctcgggt
     4861 atcgggcctc ggccaagaaa gcccaaattt gccagaaaca ggtcaagtat ctggggtatc
     4921 ttctaaaaga gggtcagaga tggctgactg aggccagaaa agagactgtg atggggcagc
     4981 ctactccgaa gacccctcga caactaaggg agttcctagg gaaggcaggc ttctgtcgcc
     5041 tcttcatccc tgggtttgca gaaatggcag cccccctgta ccctctcacc aaaccgggga
     5101 ctctgtttaa ttggggccca gaccaacaaa aggcctatca agaaatcaag caagctcttc
     5161 taactgcccc agccctgggg ttgccagatt tgactaagcc ctttgaactc tttgtcgacg
     5221 agaagcaggg ctacgccaaa ggtgtcctaa cgcaaaaact gggaccttgg cgtcggccgg
     5281 tggcctacct gtccaaaaag ctagacccag tagcagctgg gtggccccct tgcctacgga
     5341 tggtagcagc cattgccgta ctgacaaagg atgcaggcaa gctaaccatg ggacagccac
     5401 tagtcattct ggccccccat gcagtagagg cactagtcaa acaacccccc gaccgctggc
     5461 tttccaacgc ccggatgact cactatcagg ccttgctttt ggacacggac cgggtccagt
     5521 tcggaccggt ggtagccctg aacccggcta cgctgctccc actgcctgag gaagggctgc
     5581 aacacaactg ccttgatatc ctggccgaag cccacggaac ccgacccgac ctaacggacc
     5641 agccgctccc agacgccgac cacacctggt acacggatgg aagcagtctc ttacaagagg
     5701 gacagcgtaa ggcgggagct gcggtgacca ccgagaccga ggtaatctgg gctaaagccc
     5761 tgccagccgg gacatccgct cagcgggctg aactgatagc actcacccag gccctaaaga
     5821 tggcagaagg taagaagcta aatgtttata ctgatagccg ttatgctttt gctactgccc
     5881 atatccatgg agaaatatac agaaggcgtg ggtggctcac atcagaaggc aaagagatca
     5941 aaaataaaga cgagatcttg gccctactaa aagccctctt tctgcccaaa agacttagca
     6001 taatccattg tccaggacat caaaagggac acagcgccga ggctagaggc aaccggatgg
     6061 ctgaccaagc ggcccgaaag gcagccatca cagagactcc agacacctct accctcctca
     6121 tagaaaattc atcaccctct ggcggctcaa aaagaaccgc cgacggcagc gaattcgagc
     6181 ccaagaagaa gaggaaagtc ggatccggcg caacaaactt ctctctgctg aaacaagccg
     6241 gagatgtcga agagaatcct ggaccgatgg ccaagccttt gtctcaagaa gaatccaccc
     6301 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     6361 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     6421 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     6481 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     6541 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     6601 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     6661 gctaaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag cctcgactgt
     6721 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     6781 aggtgccact cccactgtcc tttcctaata aaatgagaaa attgcatcgc attgtctgag
     6841 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     6901 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     6961 cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     7021 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     7081 aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     7141 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     7261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacactca
     8041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     8101 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     8161 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     8221 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     8281 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     8341 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8401 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8461 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8521 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8581 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8641 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8701 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8761 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8821 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8881 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8941 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     9001 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     9061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     9121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     9181 agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata
     9241 gttaagccag tatctgctcc ctgcttgtgt gtt
//
