LOCUS       sequence-353573-        4482 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription of anti-CRISPR AcrIIA2 mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4482)
  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 4482)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-AcrIIA2
FEATURES             Location/Qualifiers
     source          1..4482
                     /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    534..668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(558..577)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     612..631
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(781..801)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(781..799)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(820..836)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(820..836)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(833..855)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    844..860
                     /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(868..874,875..892,893..898))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    913..934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1051..1068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1222..1810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1302..1321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1981..2772,2773..2841))
                     /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     2604..2623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2842..2946)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2972..3427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3109..3130)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3321..3340
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3498..4482
                     /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 ccatgacgct gacccgcgct cagaaaaaat acgccgaggc gatgcatgag
      181 tttatcaata tggttgatga ctttgaagaa tcaacgcctg actttgcaaa agaggttctg
      241 cacgactccg actatgtggt cattacaaaa aacgagaaat atgccgtggc actctgtagt
      301 ctctccacag atgaatgtga gtacgatact aacttgtatt tggatgaaaa gctcgtcgat
      361 tacagcacag ttgatgtcaa cggagtgaca tattacatca atatagtgga aacaaatgac
      421 atagatgatc ttgaaattgc gaccgacgag gacgagatga agtctggaaa ccaagagatt
      481 attcttaagt ccgaactgaa gtaatctaga actatagtga gtcgtattac gtagatccag
      541 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
      601 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
      661 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
      721 aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca gcttttgttc
      781 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
      841 aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc
      901 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
      961 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1021 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1081 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1141 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1201 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1261 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1321 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1381 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1441 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1501 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1561 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1621 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1681 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1741 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1801 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1861 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1921 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1981 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2041 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2101 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2161 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2221 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2281 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2341 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2401 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2461 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2521 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2581 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2641 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2701 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2761 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2821 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2881 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2941 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     3001 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3061 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3121 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3181 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3241 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3301 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3361 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3421 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3481 tcttcgctat tacgccagtc gaccgccaat tcaatatggc gtatatggac tcatgccaat
     3541 tcaatatggt ggatctggac ctgtgccaat tcaatatggc gtatatggac tcgtgccaat
     3601 tcaatatggt ggatctggac cccagccaat tcaatatggc ggacttggca ccatgccaat
     3661 tcaatatggc ggacctggca ctgtgccaac tggggagggg tctacttggc acggtgccaa
     3721 gtttgaggag gggtcttggc cctgtgccaa gtccgccata ttgaattggc atggtgccaa
     3781 taatggcggc catattggct atatgccagg atcaatatat aggcaatatc caatatggcc
     3841 ctatgccaat atggctattg gccaggttca atactatgta ttggccctat gccatatagt
     3901 attccatata tgggttttcc tattgacgta gatagcccct cccaatgggc ggtcccatat
     3961 accatatatg gggcttccta ataccgccca tagccactcc cccattgacg tcaatggtct
     4021 ctatatatgg tctttcctat tgacgtcata tgggcggtcc tattgacgta tatggcgcct
     4081 cccccattga cgtcaattac ggtaaatggc ccgcctggct caatgcccat tgacgtcaat
     4141 aggaccaccc accattgacg tcaatgggat ggctcattgc ccattcatat ccgttctcac
     4201 gccccctatt gacgtcaatg acggtaaatg gcccacttgg cagtacatca atatctatta
     4261 atagtaactt ggcaagtaca ttactattgg aagtacgcca gggtacattg gcagtactcc
     4321 cattgacgtc aatggcggta aatggcccgc gatggctgcc aagtacatcc ccattgacgt
     4381 caatggggag gggcaatgac gcaaatgggc gttccattga cgtaaatggg cggtaggcgt
     4441 gcctaatggg aggtctatat aagcaatgct cgtttaggga ac
//
