LOCUS       sequence-379672-        3987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template vector to amplify single sgRNAs or pieces for multiplexing 
            arrays. Has flanking BsaI-sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3987)
  AUTHORS   Cengic I, Canadas IC, Minton NP, Hudson EP
  TITLE     Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated 
            Single-Target Genome Editing in Synechocystis Sp. PCC 6803.
  JOURNAL   ACS Synth Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375.
  PUBMED    35969224
REFERENCE   2  (bases 1 to 3987)
  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: "ACS Synth 
            Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375."
COMMENT     Sequence Label: pMD19-PBBa_J23117-sgRNA-Spr
FEATURES             Location/Qualifiers
     source          1..3987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    433..452
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     promoter        471..505
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     misc_RNA        527..602
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      642..713
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      729..756
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(798..1589)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1590..1681)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    1705..1725
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1766..1782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1766..1782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1779..1801)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1790..1806
                     /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(1814..1820,1821..1838,1839..1844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1859..1880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1997..2014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2168..2756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2248..2267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2927..3718,3719..3787))
                     /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     3550..3569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3788..3892)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3960..3978
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat
      421 cttccagaga ttgaattcgc ggccgcttct agaacacgtg gtctcggttt ttgacagcta
      481 gctcagtcct agggattgtg ctagctacta tatttttcaa agatgagttt tagagctaga
      541 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      601 gctttttttt tcgcgagacc gacgttacta gagccaggca tcaaataaaa cgaaaggctc
      661 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga
      721 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tatactagag ccctcggctt
      781 gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt cacgtagtgg
      841 acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa
      901 gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag
      961 tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac
     1021 aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
     1081 aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc
     1141 gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga
     1201 tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct
     1261 ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca
     1321 atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
     1381 aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc
     1441 aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg
     1501 gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc
     1561 gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta ttgaagcatt
     1621 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1681 atagctagct cactcggtcg ctactactag tagcggccgc tgcagaatcg tcgaacggca
     1741 ggcgtgcaaa cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1801 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1861 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1921 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1981 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2041 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2101 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2161 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2221 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2281 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2341 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2401 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2461 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2521 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2581 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2641 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2701 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2761 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2821 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2881 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2941 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3001 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3061 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3121 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3181 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3241 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3301 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3361 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3421 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3481 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3541 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3601 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3661 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3721 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3781 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3841 gcggatacat atttgaatgt atttagaaaa ataaacaaat gggggttccg cgcacatttc
     3901 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     3961 ataggcgtat cacgaggccc tttcgtc
//
