LOCUS       sequence-373346-        5783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes lacI- and GFP-targeting gRNAs for positive and negative 
            selections of dCas9 activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5783)
  AUTHORS   Spisak S, O'Brien B, Ostermeier M
  TITLE     A bacterial dual positive and negative selection system for dCas9 
            activity.
  JOURNAL   PLoS One. 2022 Jun 3;17(6):e0269270. doi: 
            10.1371/journal.pone.0269270. eCollection 2022.
  PUBMED    35657952
REFERENCE   2  (bases 1 to 5783)
  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: "PLoS One.";
            date: "2022-06-3"; volume: "17(6):e0269270. doi"; pages: " 
            10.1371/journal.pone.0269270. eCollection 2022"
COMMENT     Sequence Label: pSelect-6
FEATURES             Location/Qualifiers
     source          1..5783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        71..146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             167..196
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             212..229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      455..501
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        537..571
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        592..667
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             688..717
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             733..750
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      976..1022
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1075..1092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1246..1834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1326..1345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2012..2103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2104..2895
                     /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"
     CDS             complement(join(3068..3859,3860..3928))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3691..3710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(3940..3962)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3951..3967
                     /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(3975..3981,3982..3997,3998..4003))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(4316..5398)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5360..5379
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5399..5476)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5716..5734
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(5738..5781)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt catctaattc
       61 aacaagaatt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      121 ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat
      181 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
      241 ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
      301 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
      361 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
      421 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
      481 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tcaattctaa agatctttga
      541 cagctagctc agtcctaggt ataatactag tgctggcctg gttcaccacg cgttttagag
      601 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      661 tcggtgcttt ttttgaagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat
      721 agcgccgtcg accatcatca tcatcatcat tgagtttaaa cggtctccag cttggctgtt
      781 ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc
      841 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
      901 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
      961 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     1021 atctgttgtt tgtcggtgaa ctggatcctt actcgagtct agactgcagg cttcctcgct
     1081 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     1141 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     1201 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc acaggctccg
     1261 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1321 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1381 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1441 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1501 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     1561 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1621 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1681 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1741 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1801 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1861 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1921 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1981 atatgagtaa acttgtacac tccgctagct atttgtttat ttttctaaat acattcaaat
     2041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2101 agtatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     2161 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     2221 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     2281 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     2341 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     2401 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     2461 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     2521 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     2581 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     2641 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     2701 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     2761 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     2821 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     2881 gtagtcggca aataatgtct aacaattcgt tcaagccgag gggccgcaag atccggccac
     2941 gatgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgctgg
     3001 tttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggt
     3061 tactagttta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3121 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3181 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3241 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3301 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3361 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3421 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3481 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3541 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3601 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3661 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     3721 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3781 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3841 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3901 gggcgacacg gaaatgttga atactcatgg atccttcctc ctgtgtgaaa ttgttatccg
     3961 ctcacaattc cacacattat acgagccgat gattaattgt caacaggggg atggggagta
     4021 agcttggccg ggctcagcac gttaccggac cagaagttgc aagattccga ataccgcaag
     4081 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4141 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4201 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4261 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4321 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4381 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4441 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4501 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4561 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4621 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4681 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4741 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4801 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4861 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4921 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4981 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5041 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5101 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5161 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5221 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5281 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5341 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcatat
     5401 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5461 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5521 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5581 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5641 gcgctcatga catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     5701 acattaacct ataaaaatag gcgtatcacg aggcagaatt tcagataaaa aaaatcctta
     5761 gctttcgcta aggatgattt ctg
//
