LOCUS       sequence-371948-        5262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes lacI-targeting gRNA for positive selection of dCas9 
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5262)
  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 5262)
  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-2
FEATURES             Location/Qualifiers
     source          1..5262
                     /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"
     primer_bind     complement(285..302)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      455..501
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(554..571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(725..1313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(805..824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1491..1582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1583..2374
                     /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(2547..3338,3339..3407))
                     /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     3170..3189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(3419..3441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3430..3446
                     /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(3454..3460,3461..3476,3477..3482))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(3795..4877)
                     /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     4839..4858
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4878..4955)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5195..5213
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(5217..5260)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt gctggcctgg
       61 ttcaccacgc 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 tggatcctta ctcgagtcta
      541 gactgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      601 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      661 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      721 cgtttttcca caggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      781 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      841 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      901 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      961 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1021 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1081 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1141 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1201 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1261 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1321 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1381 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1441 ttaaatcaat ctaaagtata tatgagtaaa cttgtacact ccgctagcta tttgtttatt
     1501 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1561 ataatattga aaaaggaaga gtatgaggga agcggtgatc gccgaagtat cgactcaact
     1621 atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt
     1681 gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac
     1741 ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac
     1801 ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca
     1861 cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca
     1921 gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat
     1981 cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact
     2041 ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg
     2101 gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat
     2161 ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat
     2221 ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg
     2281 acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt
     2341 gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acaattcgtt caagccgagg
     2401 ggccgcaaga tccggccacg atgacccggt cgtcggttca gggcagggtc gttaaatagc
     2461 cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg
     2521 cttctcaaat gcctgaggtt actagtttac caatgcttaa tcagtgaggc acctatctca
     2581 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2641 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2701 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2761 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2821 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca
     2881 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2941 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3001 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3061 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3121 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg
     3181 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3241 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3301 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3361 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatgga tccttcctcc
     3421 tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccgatg attaattgtc
     3481 aacaggggga tggggagtaa gcttggccgg gctcagcacg ttaccggacc agaagttgca
     3541 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3601 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3661 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3721 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3781 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3841 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3901 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3961 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4021 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4081 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4141 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4201 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4261 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4321 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4381 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4441 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4501 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4561 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4621 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4681 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4741 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4801 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4861 aacgttactg gtttcatatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4921 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4981 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5041 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5101 catacccacg ccgaaacaag cgctcatgac atttccccga aaagtgccac ctgacgtcta
     5161 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggcagaattt
     5221 cagataaaaa aaatccttag ctttcgctaa ggatgatttc tg
//
