LOCUS       sequence-389302-        4612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For SECRETS protocol to screen for gRNA activity and specificity: 
            BsaI cassette for gRNA expression or for cloning (x-)gRNA librarys +
            off-target for SECRETS counter-selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4612)
  AUTHORS   Herring-Nicholas A, Dimig H, Roesing MR, Josephs EA
  TITLE     Selection of extended CRISPR RNAs with enhanced targeting and 
            specificity.
  JOURNAL   Commun Biol. 2024 Jan 12;7(1):86. doi: 10.1038/s42003-024-05776-8.
  PUBMED    38212640
REFERENCE   2  (bases 1 to 4612)
  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: "Commun 
            Biol."; date: "2024-01-12"; volume: "7(1):86. doi"; pages: " 
            10.1038/s42003-024-05776-8"
COMMENT     Sequence Label: pSECRETS-B
FEATURES             Location/Qualifiers
     source          1..4612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..630)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(631..735)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    812..830
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    837..855
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        937..1012
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           1019..1086
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      1200..1271
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1287..1314
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1845..2795)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(3557..3651)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3682..4476)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3793..3812)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4403..4422
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta aacgagacat actcttcctt tttcaatatt attgaagcat
      661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcctct ggctaactag
      781 agaacccact gcttactggc ttatcgaaat ttccctatca gtgatagaga ttgacatccc
      841 tatcagtgat agagatactg agcacgtgag acccatgcca tagcgttgtt tagggataac
      901 agggtaatac tgtccacaca atctgccctg gtctccgttt tagagctaga aatagcaagt
      961 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgg
     1021 ccggcatggt cccagcctcc tcgctggcgc cggctgggca acatgcttcg gcatggcgaa
     1081 tgggacgaca tcacctccca caacgaagac tacaccatcg ttgaacagta cgaacgtgct
     1141 gaaggtcgtc actccaccgg tgcttaagga tccaaactcg agtaaggatc tccaggcatc
     1201 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     1261 tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata
     1321 cctagggtac gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc
     1381 agaatcgcag atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt
     1441 gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt
     1501 cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt
     1561 gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat
     1621 taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttga
     1681 atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg
     1741 tgcatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg
     1801 ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat
     1861 ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca
     1921 ttgagatcat acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga
     1981 atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct
     2041 gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg
     2101 gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg
     2161 cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt
     2221 ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg
     2281 aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt
     2341 tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt
     2401 tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta
     2461 aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca
     2521 aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct
     2581 ttagctaata caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta
     2641 taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt
     2701 gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct
     2761 agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt
     2821 taatcactat accaattgag atgggctagt caatgataat tactagtcct tttcccgggt
     2881 gatctgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga
     2941 ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca agtggttata
     3001 atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat
     3061 aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct
     3121 cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc
     3181 aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg
     3241 tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag
     3301 gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg
     3361 cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca
     3421 gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat
     3481 tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac
     3541 tgtccctagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga
     3601 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
     3661 tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg
     3721 ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc
     3781 aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc
     3841 cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa
     3901 gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct
     3961 ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac
     4021 aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa
     4081 tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac
     4141 tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag
     4201 cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt
     4261 cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag
     4321 gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc
     4381 agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc
     4441 cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc
     4501 ttgatcagat catgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt
     4561 tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt cc
//
