LOCUS       Exported                5189 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  AAV vector for expression of AcrIIA4 with two miR-1 binding sites.
ACCESSION   .
VERSION     .
KEYWORDS    AAV CMV-driven AcrIIA4-2xmiR-1 target sites
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5189)
  AUTHORS   Hoffmann MD, Aschenbrenner S, Grosse S, Rapti K, Domenger C, Fakhiri
            J, Mastel M, Borner K, Eils R, Grimm D, Niopek D
  TITLE     Cell-specific CRISPR-Cas9 activation by microRNA-dependent 
            expression of anti-CRISPR proteins.
  JOURNAL   Nucleic Acids Res. 2019 Apr 15. pii: 5458120. doi: 
            10.1093/nar/gkz271.
  PUBMED    30982889
REFERENCE   2  (bases 1 to 5189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5189
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             129..989
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2072..2102
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2604..2844
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2604..2624
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2775..2794
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        3000..3379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3380..3583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3534..3554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        3628..3646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3668..3691
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4105..4122)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4111..4335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(4546..4564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4574..4591)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4574..4590)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4583..4605)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4731..5186)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4868..4889)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5080..5099
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccact agtatgcatc
     2221 ctgcaggacg cgtcctaccc actccctcta tgcgcgctcg ctcactcact cggccctgga
     2281 gaccaaaggt ctccagggcc gagtgagtga gcgagcgcgc atagagggag tgggtacctc
     2341 catcatctgg tttcccagat ctgatatcgg cgcgcccgag tccaacaccc gtgggaatcc
     2401 catgggcacc atggcccctc gctccaaaaa tgctttcgcg tcgcgcagac actgctcggt
     2461 agtttcgggg atcagcgttt gagtaagagc ccgcgtctga accctccgcg ccgccccggc
     2521 cccagtggaa agacgcgcag gcaaaacgca ccacgtgacg gagcgtgacc gcgcgccgag
     2581 cgcgcgccaa ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata
     2641 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     2701 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     2761 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     2821 ctttatatat cttgtggaaa ggacgaaaca ccgagtcttc gaggaagaca ggtttaagag
     2881 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2941 tggcaccgag tcggtgcttt ttttgcggcc gcggccgctc tacaaatgtg gtatctagag
     3001 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     3061 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     3121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     3181 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     3241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     3301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3361 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3421 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3481 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3541 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     3601 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     3661 ctagatggac tataaggacg atgacgataa agctggcggc agcatgaaca ttaacgacct
     3721 gattcgggaa ataaagaaca aggactacac ggtcaagctg tcaggcaccg actccaactc
     3781 tataacacag ctcattataa gggttaataa tgatggcaac gaatatgtca tctctgaatc
     3841 cgagaatgaa tcaatcgtgg agaaattcat cagtgcattt aaaaacggct ggaaccagga
     3901 gtacgaagat gaggaagagt tctacaatga tatgcaaact atcacactga aatctgagct
     3961 gaactagctc gagcggccgc cactgtgctg gatatctgca gaattataca tacttcttta
     4021 cattccatta tatatacata cttctttaca ttccattaat tagagacgag ctcgtctcta
     4081 gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     4141 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     4201 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     4261 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     4321 cggtgggctc tatgggtcga ctcgacgata tcagatctga ggaaccccta gtgatggagt
     4381 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4441 gacgcccggg ctttgcccgg gcggcctcag tgagcgagsg agcgsgcagr srgagagggg
     4501 gggscccccc cccccccccc kgcattctag agagctccaa ttcgccctat agtgagtcgt
     4561 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4621 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4681 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
     4741 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4801 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     4861 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     4921 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     4981 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5041 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5101 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5161 gcttaatgcg ccgctacagg gcgcgtcag
//
