LOCUS       sequence-396686-        4968 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Backbone for cloning longer RNA triggers that activate iSBH-sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4968)
  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 4968)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-ModFlanks
FEATURES             Location/Qualifiers
     source          1..4968
                     /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      718..1146
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(805..824)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1015..1036
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1155..1175)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1160..1489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1340..1475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1402..1421
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1536..2495
                     /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    2641..2762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2678..2697)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2732..2751
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2811..2827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2811..2827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2824..2846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2835..2851
                     /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(2859..2865,2866..2883,2884..2889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2904..2925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3042..3059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3213..3801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3293..3312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3972..4763,4764..4832))
                     /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     4595..4614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4833..4937)
                     /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 aagagccact gagacgtaag actcgagcgg
      541 tccgtctcag tcagggtctt cgagcattgg aagtctagat acctgcttta cgaagacctg
      601 cggccgcatg ctagtacgaa agtactagct tttttgctag caggcatgct ggggatgcgg
      661 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
      721 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      781 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      841 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      901 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      961 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1021 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1081 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1141 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     1201 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     1261 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     1321 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     1381 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     1441 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     1501 agcttgtata tccattttcg gatctgatcg ccaccatgtc ggtaccgctg actacctcag
     1561 cattcggcca cgcgtttctg gctaactgtg aacgcgagca gatccacctg gcgggctcca
     1621 ttcagccgca cggtatcctg ctggctgtga aagagccgga caacgtggtg atccaggctt
     1681 ctattaacgc tgcggagttc ctgaacacca actctgttgt tggccgtccg ctgcgtgacc
     1741 tgggcggcga tctgcctttg cagatcctgc cgcacctgaa cggcccgctg cacctggctc
     1801 cgatgaccct gcgttgtacc gtgggttctc cgccgcgtcg tgtggactgt accattcatc
     1861 gtccgtctaa cggcggcctg atcgtagaac tggaaccagc aaccaagacc actaacattg
     1921 cgccggctct ggacggtgcg tttcatcgta tcacttcttc atcctccctg atgggcctgt
     1981 gtgacgaaac cgcgactatt atccgtgaga ttactggcta cgaccgtgtg atggtagtac
     2041 gtttcgatga agagggtaat ggccaaattc tgtccgaacg tcgtcgtgcg gacctggaag
     2101 cgttcctggg taaccgctac ccggcgtcta ctattccgca gatcgctcgt cgcctgtacg
     2161 aacataaccg tgttcgcctg ctggtagatg tgaactatac tccggttccg ctacagccgc
     2221 gcatcagccc gctgaacggt cgtgatctgg atatgtccct gtcttgcctg cgctctatgt
     2281 ccccgtgcca ccagaaatac atgcaggaca tgggcgttgg cgcgaccctg gtttgctctc
     2341 tgatggtgtc tggtcgtctg tggggtctga tcgcttgcca ccactacgaa ccgcgcttcg
     2401 ttccgttcca cattcgcgct gctggcgaag cgctggcgga aacttgtgcg atccgcatcg
     2461 cgacgctgga gagctttgca cagtctcagt ccaaatgatg tacaagtaaa gcacgtgcta
     2521 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     2581 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     2641 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2701 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2761 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     2821 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2881 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2941 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3001 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3061 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3121 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3181 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3241 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3301 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3361 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3421 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3481 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3541 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3601 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3661 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3721 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3781 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3841 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3901 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3961 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4021 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4081 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4141 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4201 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4261 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4321 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4381 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4441 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4501 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4561 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4621 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4681 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4741 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4801 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4861 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4921 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
