LOCUS       sequence-396676-        4926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning short RNA triggers that activate iSBH-sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4926)
  AUTHORS   Pelea O, Mayes S, Ferry QRV, Fulga TA, Sauka-Spengler T
  TITLE     Specific Modulation of CRISPR Transcriptional Activators through 
            RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos
  JOURNAL   bioRxiv 2023.05.08.539738
REFERENCE   2  (bases 1 to 4926)
  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: "bioRxiv 
            2023.05.08.539738"
COMMENT     Sequence Label: U6-TM2Emp-6T_iBlue
FEATURES             Location/Qualifiers
     source          1..4926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        255..503
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     255..275
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     426..445
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     rep_origin      676..1104
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(763..782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     973..994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1113..1133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1118..1447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1298..1433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1360..1379
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1494..2453
                     /codon_start=1
                     /product="naturally monomeric infrared fluorescent protein 
                     derived from a truncated bacteriophytochrome (Yu et al., 
                     2015)"
                     /label=mIFP
                     /translation="MSVPLTTSAFGHAFLANCEREQIHLAGSIQPHGILLAVKEPDNVV
                     IQASINAAEFLNTNSVVGRPLRDLGGDLPLQILPHLNGPLHLAPMTLRCTVGSPPRRVD
                     CTIHRPSNGGLIVELEPATKTTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYD
                     RVMVVRFDEEGNGQILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYT
                     PVPLQPRISPLNGRDLDMSLSCLRSMSPCHQKYMQDMGVGATLVCSLMVSGRLWGLIAC
                     HHYEPRFVPFHIRAAGEALAETCAIRIATLESFAQSQSK"
     polyA_signal    2599..2720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2636..2655)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2690..2709
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2769..2785)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2769..2785)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2782..2804)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2793..2809
                     /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(2817..2823,2824..2841,2842..2847))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2862..2883
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3000..3017)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3171..3759)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3251..3270)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3930..4721,4722..4790))
                     /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     4553..4572
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4791..4895)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagtgagggc ctatttccca tgattccttc atatttgcat atacgataca
      301 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      481 tcttgtggaa aggacgaaac accggctcga aagagccacg ggtcttcgag cattggaagt
      541 ctagatacct gctttacgaa gacctatgct agtacgaaag tactagcttt tttgctagca
      601 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
      661 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      721 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1021 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1081 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     1141 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1201 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1261 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1321 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1381 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1441 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcgcc accatgtcgg
     1501 taccgctgac tacctcagca ttcggccacg cgtttctggc taactgtgaa cgcgagcaga
     1561 tccacctggc gggctccatt cagccgcacg gtatcctgct ggctgtgaaa gagccggaca
     1621 acgtggtgat ccaggcttct attaacgctg cggagttcct gaacaccaac tctgttgttg
     1681 gccgtccgct gcgtgacctg ggcggcgatc tgcctttgca gatcctgccg cacctgaacg
     1741 gcccgctgca cctggctccg atgaccctgc gttgtaccgt gggttctccg ccgcgtcgtg
     1801 tggactgtac cattcatcgt ccgtctaacg gcggcctgat cgtagaactg gaaccagcaa
     1861 ccaagaccac taacattgcg ccggctctgg acggtgcgtt tcatcgtatc acttcttcat
     1921 cctccctgat gggcctgtgt gacgaaaccg cgactattat ccgtgagatt actggctacg
     1981 accgtgtgat ggtagtacgt ttcgatgaag agggtaatgg ccaaattctg tccgaacgtc
     2041 gtcgtgcgga cctggaagcg ttcctgggta accgctaccc ggcgtctact attccgcaga
     2101 tcgctcgtcg cctgtacgaa cataaccgtg ttcgcctgct ggtagatgtg aactatactc
     2161 cggttccgct acagccgcgc atcagcccgc tgaacggtcg tgatctggat atgtccctgt
     2221 cttgcctgcg ctctatgtcc ccgtgccacc agaaatacat gcaggacatg ggcgttggcg
     2281 cgaccctggt ttgctctctg atggtgtctg gtcgtctgtg gggtctgatc gcttgccacc
     2341 actacgaacc gcgcttcgtt ccgttccaca ttcgcgctgc tggcgaagcg ctggcggaaa
     2401 cttgtgcgat ccgcatcgcg acgctggaga gctttgcaca gtctcagtcc aaatgatgta
     2461 caagtaaagc acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     2521 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     2581 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2641 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2701 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     2761 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2821 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2881 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2941 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3001 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3061 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3121 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3181 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3241 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3301 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3361 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3421 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3481 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3541 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3601 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3661 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3721 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3781 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3841 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3901 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3961 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4021 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4081 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4141 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4201 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4261 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4321 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4381 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4441 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4501 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4561 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4621 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4681 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4741 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4801 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4861 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4921 gacgtc
//
