LOCUS       Exported                5792 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  mamalian expression of AcrIIA2.
ACCESSION   .
VERSION     .
KEYWORDS    pJH373
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)
  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."
FEATURES             Location/Qualifiers
     source          1..5792
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     670..689
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        670..688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1190..1207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1196..1420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1466..1894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1553..1572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1763..1784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1903..1923)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1908..2237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2088..2223
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2150..2169
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2304..3098
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2358..2377)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2968..2987
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3272..3393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3309..3328)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3363..3382
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3442..3458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3442..3458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3455..3477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3466..3482
                     /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(3490..3520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3535..3556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /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"
     primer_bind     complement(3924..3943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4603..5463)
                     /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     5226..5245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5464..5568)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5643..5662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"