LOCUS       sequence-435309-        7740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a sgRNA template with a protospacer cloning site flanked 
            by BsaI restriction sites for easy insertion of a targeting 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7740)
  AUTHORS   Gopalakrishna KP, Hillebrand GH, Bhavana VH, Elder JL, D'Mello A, 
            Tettelin H, Hooven TA
  TITLE     Group B Streptococcus Cas9 variants provide insight into 
            programmable gene repression and CRISPR-Cas transcriptional effects.
  JOURNAL   Commun Biol. 2023 Jun 9;6(1):620. doi: 10.1038/s42003-023-04994-w.
  PUBMED    37296208
REFERENCE   2  (bases 1 to 7740)
  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: "2023-06-9"; volume: "6(1):620. doi"; pages: " 
            10.1038/s42003-023-04994-w"
COMMENT     Sequence Label: p3015b
FEATURES             Location/Qualifiers
     source          1..7740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    813..831
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     primer_bind     complement(1022..1039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1193..1781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1273..1292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             2034..2042
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2050..2673
                     /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     complement(2123..2142)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             4465..5175
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4610..4628)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4878..4897
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5209..5226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5209..5225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5218..5240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccaggaagaa atggtggcat tcaacttgct agtgttaaat cattgttgct atcgatcatt
       61 aaagtaaaaa aagaagaaaa agaaagctat ataaaggcgc tgacaaattc ttttgactta
      121 gagcatacat tcattcaaga gactttaaac aagctagcag aacgccctaa aacggacaca
      181 caactcgatt tgtttagcta tgatacaggc tgaaaataaa acccgcacta tgccattaca
      241 tttatatcta tgatacgtgt ttgttttttc tttgctgttt agcgaatgat tagcagaaat
      301 atacagagta agattttaat taattattag ggggagaagg agagagtagc ccgaaaactt
      361 ttagttggct tggactgaac gaagtgaggg aaaggctact aaaacgtcga ggggcagtga
      421 gagcgaagcg aacacttgat tttttaattt tctatctttt ataggtcatt agagtatact
      481 tatttgtcct ataaactatt tagcagcata atagatttat tgaataggtc atttaagttg
      541 agcatattag aggaggaaaa tcttggagaa atatttgaag aacccgatta catggattgg
      601 attagttctt gtggttacgt ggtttttaac taaaagtagt gaatttttga tttttggtgt
      661 gtgtgtcttg ttgttagtat ttgctagtca aagtgattaa atagaattct catgtttgac
      721 agcttatcat cggagctccg atgataagct gtcaaacatg agaattcccg gggatcctgt
      781 cgagttcatg aaaaactaaa aaaaatattg acactctatc attgatagag tataattaaa
      841 atagagaccg cgtactacgc ctattggtct cgttttagag ctgtgaaaca gcacagcacg
      901 ttaaaataag gcagtgattt ttaatccagt ccgtattcag cttgaaaaag tgagcaccga
      961 atcggtgctt tttttctaga gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1021 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1081 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1141 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1201 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1261 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1321 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1381 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1441 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1501 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1561 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1621 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1681 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1741 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1801 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1861 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1921 aaagtatata tgagtaaact tggtctgaca gccctcgagc ataaggctcg taggctatat
     1981 tcagggcgac cacaacggtt tccctctaca aataattttg tttaactttg aaataaggag
     2041 gtaatacaaa tgtctcgttt agataaaagt aaagtgatta acagcgcatt agagctgctt
     2101 aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag
     2161 cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt
     2221 gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat
     2281 tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca
     2341 aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta
     2401 gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgctgtg
     2461 gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa
     2521 agggaaacac ctactactga tagtatgccg ccattattac gacaagctat cgaattattt
     2581 gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta
     2641 gaaaaacaac ttaaatgtga aagtgggtct taactcgagc caatcgatac gattttgaag
     2701 tggcaacaga taaaaaaaag cagtttaaaa ttgttgctga acttttaaaa caagcaaata
     2761 caatcattgt cgcaacagat agcgacagag aaggcgaaaa cattgcctgg tcgatcattc
     2821 ataaagcaaa tgccttttct aaagataaaa cgtataaaag actatggatc aatagtttag
     2881 aaaaagatgt gatccgtagc ggttttcaaa atttgcgacc aggaatgaat tactatccct
     2941 tttatcaaga agcgcacaaa aagaaaaacg aaatgataca ccaatcagtg caaaaaaaga
     3001 tataatggga gataagacgg ttcgtgttcg tgctgacttg caccatatca taaaaatcga
     3061 aacagcaaag aatggcggaa acgtaaaaga agttatggaa ataagactta gaagcaaact
     3121 taagagtgtg ttgatagtgc agtatcttaa aattttgtat aataggaatt gaagttaaat
     3181 tagatgctaa aaatttgtaa ttaagaagga gtgattacat gaacaaaaat ataaaatatt
     3241 ctcaaaactt tttaacgagt gaaaaagtac tcaaccaaat aataaaacaa ttgaatttaa
     3301 aagaaaccga taccgtttac gaaattggaa caggtaaagg gcatttaacg acgaaactgg
     3361 ctaaaataag taaacaggta acgtctattg aattagacag tcatctattc aacttatcgt
     3421 cagaaaaatt aaaactgaat actcgtgtca ctttaattca ccaagatatt ctacagtttc
     3481 aattccctaa caaacagagg tataaaattg ttgggagtat tccttaccat ttaagcacac
     3541 aaattattaa aaaagtggtt tttgaaagcc atgcgtctga catctatctg attgttgaag
     3601 aaggattcta caagcgtacc ttggatattc accgaacact agggttgctc ttgcacactc
     3661 aagtctcgat tcagcaattg cttaagctgc cagcggaatg ctttcatcct aaaccaaaag
     3721 taaacagtgt cttaataaaa cttacccgcc ataccacaga tgttccagat aaatattgga
     3781 agctatatac gtactttgtt tcaaaatggg tcaatcgaga atatcgtcaa ctgtttacta
     3841 aaaatcagtt tcatcaagca atgaaacacg ccaaagtaaa caatttaagt accgttactt
     3901 atgagcaagt attgtctatt tttaatagtt atctattatt taacgggagg aaataattct
     3961 atgagtcgct tttgtaaatt tggaaagtta cacgttacta aagggaatgt agataaatta
     4021 ttaggtatac tactgacagc ttccaaggag ctaaagagat ctaaaatagt acgtttctta
     4081 ttaaatttat tttttaagaa aaacattagg ctaacctatt tgataaaaat agtgataagt
     4141 agaaattttc taaaatattt agatttatat aaggtggtaa attatcgtta tggtttttta
     4201 catgaaaatc taaattaaat tatcagaaat taacaaaagg ataggcgcaa gtatattata
     4261 gcaaaatgat aatattagtt ttatgaaaaa aatgcagttt atttgtaata atgtaaaata
     4321 taggggaaaa gaaagcgctt gacgaccttt tggacaagta gtaagataca aacatgggcc
     4381 ctgtaaatta aaaatactgc agtaaaagtg attttagttt aaaggaggaa atttattatg
     4441 aacgttaaac atatgatgga tcccatggtg agcaagggcg aggaggataa catggccatc
     4501 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     4561 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     4621 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     4681 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     4741 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     4801 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     4861 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     4921 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     4981 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     5041 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     5101 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     5161 gagctgtaca agtaatctag agtcgacctg caggcatgca agcttggcac tggccgtcgt
     5221 tttacaacgt cgtgactggg aaaaccctgg cctagcgctt agaatcgctt taggaaacac
     5281 gatccagtcc aataatcgtc gataaaaact tttgaaaaag gttggtgaaa ttacctactt
     5341 ttggaataat cacaaatcac aagtgattaa tcacaaatca caagtgatta atcacttgtt
     5401 tattaagata ttaaaagcta taatttaaat aaagcgtgaa ttttattaca caaaaagagg
     5461 ggggagaaac ttggaactag catttagaga aagcttaaaa aagatgagag gtaccaaatc
     5521 aaaagaaaaa ttctcccaag aattagaaat gagtagatca aattattcac gaatagaatc
     5581 aggaaaatca gatccaacca taaaaacact agaacaaatt gcaaagttaa ctaactcaac
     5641 gctagtagtg gatttaatcc caaatgagcc aacagaacca gaaccagaaa cagaatcaga
     5701 acaagtaaca ttggatttag aaatggaaga agaaaaaagc aatgacttcg tgtgaataat
     5761 gcgcgaaatc gttgcttatt ttttttaaaa gcggtatact agatataacg aaacaacgaa
     5821 ctgaatagaa acgaaaaaag agccatgaca catttataaa atgtttgacg acattttata
     5881 aatgcatagc ccgataagat tgccaaacca acgcttatca gttagtcaga tgaactcttc
     5941 cctcgtaaga agttatttaa ttaactttgt ttgaagacgg tatataaccg tactatcatt
     6001 atatagggaa atcagagagt tttcaagtat ctaagctact gaatttaaga attgttaagc
     6061 aatcaatcgg aaatcgtttg attgcttttt ttgtattcat ttatagaagg tggagtttgt
     6121 atgaatcatg atgaatgtaa aacttatata aaaaatagtt tattggagat aagaaaatta
     6181 gcaaatatct atacactaga aacgtttaag aaagagttag aaaagagaaa tatctactta
     6241 gaaacaaaat cagataagta tttttcttcg gagggggaag attatatata taagttaata
     6301 gaaaataaca aaataattta ttcgattagt ggaaaaaaat tgacttataa aggaaaaaaa
     6361 tctttttcaa aacatgcaat attgaaacag ttgaatgaaa aagcaaacca agttaattaa
     6421 acaacctatt ttataggatt tataggaaag gagaacagct gaatgaatat cccttttgtt
     6481 gtagaaactg tgcttcatga cggcttgtta aagtacaaat ttaaaaatag taaaattcgc
     6541 tcaatcacta ccaagccagg taaaagcaaa ggggctattt ttgcgtatcg ctcaaaatca
     6601 agcatgattg gcggtcgtgg tgttgttctg acttccgagg aagcgattca agaaaatcaa
     6661 gatacattta cacattggac acccaacgtt tatcgttatg gaacgtatgc agacgaaaac
     6721 cgttcataca cgaaaggaca ttctgaaaac aatttaagac aaatcaatac cttctttatt
     6781 gattttgata ttcacacggc aaaagaaact atttcagcaa gcgatatttt aacaaccgct
     6841 attgatttag gttttatgcc tactatgatt atcaaatctg ataaaggtta tcaagcatat
     6901 tttgttttag aaacgccagt ctatgtgact tcaaaatcag aatttaaatc tgtcaaagca
     6961 gccaaaataa tttcgcaaaa tatccgagaa tattttggaa agtctttgcc agttgatcta
     7021 acgtgtaatc attttggtat tgctcgcata ccaagaacgg acaatgtaga attttttgat
     7081 cctaattacc gttattcttt caaagaatgg caagattggt ctttcaaaca aacagataat
     7141 aagggcttta ctcgttcaag tctaacggtt ttaagcggta cagaaggcaa aaaacaagta
     7201 gatgaaccct ggtttaatct cttattgcac gaaacgaaat tttcaggaga aaagggttta
     7261 atagggcgta ataacgtcat gtttaccctc tctttagcct actttagttc aggctattca
     7321 atcgaaacgt gcgaatataa tatgtttgag tttaataatc gattagatca acccttagaa
     7381 gaaaaagaag taatcaaaat tgttagaagt gcctattcag aaaactatca aggggctaat
     7441 agggaataca ttaccattct ttgcaaagct tgggtatcaa gtgatttaac cagtaaagat
     7501 ttatttgtcc gtcaagggtg gtttaaattc aagaaaaaaa gaagcgaacg tcaacgtgtt
     7561 catttgtcag aatggaaaga agatttaatg gcttatatta gcgaaaaaag cgatgtatac
     7621 aagccttatt tagtgacgac caaaaaagag attagagaag tgctaggcat tcctgaacgg
     7681 acattagata aattgctgaa ggtactgaag gcgaatcagg aaattttctt taagattaaa
//
