LOCUS       sequence-172912-        5795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mamalian expression of anti-CRISPR protein AcrIIA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5795)
  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 5795)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
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     Sequence Label: pJH375
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        407..610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     561..581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     655..674
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        655..673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1178..1195)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1184..1408
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1454..1882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1541..1560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1751..1772
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1891..1911)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1896..2225
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2076..2211
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2138..2157
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2292..3086
                     /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(2346..2365)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2956..2975
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3260..3381
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3297..3316)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3351..3370
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3430..3446)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3430..3446)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3443..3465)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3454..3470
                     /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(3478..3484,3485..3502,3503..3508))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3523..3544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3661..3678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3832..4420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3912..3931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4591..5382,5383..5451))
                     /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     5214..5233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5452..5556)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5631..5650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
       61 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      361 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      421 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      481 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      541 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      601 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      661 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg taccgagctc
      721 ggatccgcca ccatgacaaa gtataataag tcagagataa tgaaaaacgc atgggcgatg
      781 tttaacagct atgaatggga tgttgaaaat tttaaattcg tgagtgctga gaacaagacc
      841 ttctctaatt gcctcaagga agcctgggca gaagagaagg aatatgtcga gaggaaagcc
      901 aaagaaacag ccgaggcgcc ccggtctgag gaggctaaag catgggactg ggcgtgcaga
      961 aaattgaatg taaacgattt gcagaacatt gacgccactg acaaagtctt ctatgtggtt
     1021 gacatgcaaa aagaaatgtg gacttccaat gtgtgggcgc aggcaatcaa ggctgtagaa
     1081 ctgtatgtga agctcggtct tgcttaagaa ttctgcagat atccagcaca gtggcggccg
     1141 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     1201 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1261 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1321 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1381 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     1441 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1501 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1561 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1621 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1681 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1741 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1801 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1861 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     1921 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1981 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2041 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2101 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     2161 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2221 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     2281 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     2341 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     2401 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     2461 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     2521 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     2581 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     2641 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     2701 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     2761 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     2821 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2881 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     2941 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3001 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3061 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     3121 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3181 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3241 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3301 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3361 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3421 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3481 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3541 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3601 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3661 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3721 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3781 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3841 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3901 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3961 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4021 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4081 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4141 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4201 tagcagagcg aggtatgtag gcagtgctac agagttcttg aagtggtggc ctaactacgg
     4261 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4321 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4381 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4441 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4501 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4561 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4621 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4681 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4741 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4801 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4861 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4921 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4981 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5041 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5101 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5161 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5221 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5281 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5341 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5401 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5461 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5521 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5581 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     5641 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     5701 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     5761 aatctgctta gggttaggcg ttttgcgctg cttcg
//
