LOCUS       sequence-353574-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of anti-CRISPR AcrIIA4 mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4374)
  AUTHORS   Takasugi PR, Wang S, Truong KT, Drage EP, Kanishka SN, Higbee MA, 
            Bamidele N, Ojelabi O, Sontheimer EJ, Gagnon JA
  TITLE     Orthogonal CRISPR-Cas tools for genome editing, inhibition, and 
            CRISPR recording in zebrafish embryos.
  JOURNAL   Genetics. 2021 Nov 4. pii: 6420709. doi: 10.1093/genetics/iyab196.
  PUBMED    34735006
REFERENCE   2  (bases 1 to 4374)
  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: "Genetics. 
            2021 Nov 4. pii: 6420709. doi: 10.1093/genetics/iyab196."
COMMENT     Sequence Label: pCS2-AcrIIA4
FEATURES             Location/Qualifiers
     source          1..4374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             103..123
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    426..560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(450..469)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     504..523
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(673..693)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(673..691)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(712..728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(712..728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(725..747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    736..752
                     /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(760..766,767..784,785..790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    805..826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(943..960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1114..1702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1194..1213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1873..2664,2665..2733))
                     /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     2496..2515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2734..2838)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2864..3319)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3001..3022)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3213..3232
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3390..4374
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca tgccaaaaaa gaagagaaag
      121 gtagaagacc ccatgaacat taacgacctc atacgagaga ttaagaacaa agattacacc
      181 gtcaaactgt caggaactga tagtaactca atcacccagc ttattatcag ggtaaacaat
      241 gatgggaatg aatatgtgat atctgagagc gaaaacgagt ctatcgtcga gaaattcatt
      301 tccgctttta agaacgggtg gaatcaggaa tatgaggatg aagaagaatt ttacaatgac
      361 atgcagacga tcacgttgaa aagtgaactg aactaatcta gaactatagt gagtcgtatt
      421 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
      481 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
      541 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
      601 gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc
      661 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
      721 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
      781 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      841 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      901 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      961 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1021 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1081 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1141 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1201 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1261 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1321 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1381 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1441 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1501 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1561 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1621 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1681 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1741 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1801 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1861 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1921 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1981 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2041 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2101 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2161 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2221 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2281 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2341 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2401 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2461 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2521 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2581 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2641 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2701 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2761 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2821 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     2881 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2941 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3001 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3061 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3121 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3181 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3241 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3301 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     3361 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
     3421 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
     3481 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
     3541 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
     3601 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
     3661 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
     3721 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
     3781 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
     3841 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
     3901 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
     3961 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
     4021 attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat
     4081 atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat
     4141 caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat
     4201 tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat
     4261 ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg
     4321 ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaac
//
