LOCUS       40924_26111        5792 bp DNA     circular SYN 13-MAY-2021
DEFINITION  mamalian expression of AcrIIA2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  AUTHORS   Rauch BJ, Silvis MR, Hultquist JF, Waters CS, McGregor MJ, Krogan 
            NJ, Bondy-Denomy J
  TITLE     Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.
  JOURNAL   Cell. 2016 Dec 22. pii: S0092-8674(16)31683-X. doi: 
            10.1016/j.cell.2016.12.009.
  PUBMED    28041849
REFERENCE   2  (bases 1 to 5792)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5792)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2016 
            Dec 22. pii: S0092-8674(16)31683-X. doi: 
            10.1016/j.cell.2016.12.009."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        670..688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1196..1420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1466..1894
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1908..2237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2304..3095
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3272..3405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3442..3458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3466..3482)
                     /label=lac operator
                     /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(3490..3520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3535..3556)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(3673..3690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3844..4432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4606..5463)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5464..5568)
                     /label=AmpR promoter
     primer_bind     complement(5643..5662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
      661 tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag
      721 cttggtaccg agctcggatc cgccaccatg acgctgaccc gcgctcagaa aaaatacgcc
      781 gaggcgatgc atgagtttat caatatggtt gatgactttg aagaatcaac gcctgacttt
      841 gcaaaagagg ttctgcacga ctccgactat gtggtcatta caaaaaacga gaaatatgcc
      901 gtggcactct gtagtctctc cacagatgaa tgtgagtacg atactaactt gtatttggat
      961 gaaaagctcg tcgattacag cacagttgat gtcaacggag tgacatatta catcaatata
     1021 gtggaaacaa atgacataga tgatcttgaa attgcgaccg acgaggacga gatgaagtct
     1081 ggaaaccaag agattattct taagtccgaa ctgaagtaag aattctgcag atatccagca
     1141 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1201 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1261 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1321 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1381 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1441 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1501 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1561 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1621 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1681 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1741 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1801 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1861 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     1921 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1981 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2041 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2101 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2161 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2221 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2281 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2341 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2401 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2461 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2521 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     2581 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     2641 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     2701 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     2761 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     2821 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     2881 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     2941 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3001 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3061 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3121 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3181 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3241 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3301 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3361 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3421 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3481 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3541 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3601 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3661 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3721 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3781 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3841 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3901 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3961 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4021 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4081 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4141 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4201 tggtaacagg attagcagag cgaggtatgt aggcagtgct acagagttct tgaagtggtg
     4261 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4321 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4381 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4441 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4501 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4561 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4621 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4681 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4741 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4801 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4861 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4921 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4981 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5041 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5101 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5161 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5221 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5281 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5341 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5401 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5461 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5521 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5581 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     5641 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     5701 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     5761 aattgcatga agaatctgct tagggttagg cg
//
