LOCUS       sequence-137435-        2779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  self-killing gRNA plasmid pMAZ-SK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2736 to 2770)
  AUTHORS   Ronda C, Pedersen LE, Sommer MO, Nielsen AT
  TITLE     CRMAGE: CRISPR Optimized MAGE Recombineering.
  JOURNAL   Sci Rep. 2016 Jan 22;6:19452. doi: 10.1038/srep19452.
  PUBMED    26797514
REFERENCE   2  (bases 1 to 2779)
  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: "Sci Rep."; 
            date: "2016-01-22"; volume: "6:19452. doi"; pages: " 
            10.1038/srep19452"
COMMENT     Sequence Label: pMAZ-SK
FEATURES             Location/Qualifiers
     source          1..2779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   128..163
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   194..229
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   260..295
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    369..387
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    394..412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        443..518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(557..575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      571..618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(848..1663)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(848..1663)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1571..1590
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1664..1755)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1773..2408)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(1918..1937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(2657..2735)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(2736..2770)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
ORIGIN
        1 atataccatg gccacaattc agcaaattgt gaacatcatc acgttcatct ttccctggtt
       61 gccaatggcc cattttcctg tcagtaacga gaaggtcgcg aattcaggcg ctttttagac
      121 tggtcgtgtt ttagagctat gctgttttga atggtcccaa aacccgttcc gtgtagacag
      181 ttcgctccaa gctgttttag agctatgctg ttttgaatgg tcccaaaacg ccatgtttca
      241 gaaacaactc tggcgcatcg ttttagagct atgctgtttt gaatggtccc aaaacaaaaa
      301 aaaaccccgc ccctgacagg gcggggtttt ttttttaatt aaagtctggt cctcgagtct
      361 ggttataatc cctatcagtg atagagattg acatccctat cagtgataga gatactgagc
      421 acaacgaaac cgtcgttgta gtgttttaga gctagaaata gcaagttaaa ataaggctag
      481 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttatgcag cctaccggta
      541 tcctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      601 gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
      661 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
      721 cctgaaccga cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt
      781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
      841 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
      901 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
      961 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1021 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1081 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     1141 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     1201 gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag
     1261 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1321 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1381 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1441 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1501 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1561 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1621 atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat
     1681 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1741 agaaaaataa acaaataggc atgctagcgc agaaacgtcc tagaagatgc caggaggata
     1801 cttagcagag agacaataag gccggagcga agccgttttt ccataggctc cgcccccctg
     1861 acgaacatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     1921 gataccaggc gtttccccct gatggctccc tcttgcgctc tcctgttccc gtcctgcggc
     1981 gtccgtgttg tggtggaggc tttacccaaa tcaccacgtc ccgttccgtg tagacagttc
     2041 gctccaagct gggctgtgtg caagaacccc ccgttcagcc cgactgctgc gccttatccg
     2101 gtaactatca tcttgagtcc aacccggaaa gacacgacaa aacgccactg gcagcagcca
     2161 ttggtaactg agaattagtg gatttagata tcgagagtct tgaagtggtg gcctaacaga
     2221 ggctacactg aaaggacagt atttggtatc tgcgctccac taaagccagt taccaggtta
     2281 agcagttccc caactgactt aaccttcgat caaaccgcct ccccaggcgg ttttttcgtt
     2341 tacagagcag gagattacga cgatcgtaaa aggatctcaa gaagatcctt tacggattcc
     2401 cgacaccatc actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     2461 gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga
     2521 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     2581 ctaacttaca ttaattgcgt tgcgcacacc gactagcgaa aaaaccccgc cgaagcgggg
     2641 ttttttgcga aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     2701 ttattttaac ttgctatgct gttttgaatg gttccgctag cactgtacct aggactgagc
     2761 tagccgtcaa aagcagcgt
//
