LOCUS       sequence-172913-        5684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mamalian expression of anti-CRISPR protein AcrIIA4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5684)
  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 5684)
  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: pJH376
FEATURES             Location/Qualifiers
     source          1..5684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     653..672
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        653..671
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1065..1082)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1071..1295
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1341..1769
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1428..1447)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1638..1659
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1778..1798)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1783..2112
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1963..2098
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2025..2044
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2179..2973
                     /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(2233..2252)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2843..2862
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3147..3268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3184..3203)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3238..3257
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3317..3333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3317..3333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3330..3352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3341..3357
                     /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(3365..3371,3372..3389,3390..3395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3410..3431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3548..3565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3719..4307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3799..3818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4478..5269,5270..5338))
                     /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     5101..5120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5339..5443)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5518..5537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      661 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttggta ccgagctcgg
      721 atccgccacc atgaacatta acgacctcat acgagagatt aagaacaaag attacaccgt
      781 caaactgtca ggaactgata gtaactcaat cacccagctt attatcaggg taaacaatga
      841 tgggaatgaa tatgtgatat ctgagagcga aaacgagtct atcgtcgaga aattcatttc
      901 cgcttttaag aacgggtgga atcaggaata tgaggatgaa gaagaatttt acaatgacat
      961 gcagacgatc acgttgaaaa gtgaactgaa ctaagaattc tgcagatatc cagcacagtg
     1021 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     1081 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1141 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1201 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     1261 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1321 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1381 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1441 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1501 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1561 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1621 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1681 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1741 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     1801 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     1861 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1921 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     1981 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2041 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2101 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     2161 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2221 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2281 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2341 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     2401 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2461 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2521 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2581 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2641 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2701 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2761 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2821 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2881 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     2941 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3001 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3061 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3121 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3181 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3241 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3301 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3361 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3421 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3481 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3541 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3601 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3661 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3721 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3781 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3841 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3901 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3961 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4021 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4081 acaggattag cagagcgagg tatgtaggca gtgctacaga gttcttgaag tggtggccta
     4141 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4201 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4261 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4321 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4381 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4441 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4501 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4561 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4621 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4681 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4741 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4801 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4861 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4921 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4981 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5041 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5101 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5161 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5221 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5281 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5341 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5401 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5461 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     5521 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5581 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     5641 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcg
//
