LOCUS       sequence-394548-        6454 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses a Cas7-11 crRNA and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6454)
  AUTHORS   Strecker J, Demircioglu FE, Li D, Faure G, Wilkinson ME, Gootenberg 
            JS, Abudayyeh OO, Nishimasu H, Macrae RK, Zhang F
  TITLE     RNA-activated protein cleavage with a CRISPR-associated 
            endopeptidase.
  JOURNAL   Science. 2022 Nov 25;378(6622):874-881. doi: 
            10.1126/science.add7450. Epub 2022 Nov 3.
  PUBMED    36423276
REFERENCE   2  (bases 1 to 6454)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.add7450"; journalName: "Science"; date: 
            "2022-11-25- 25"; volume: "378"; issue: "6622"; pages: "874-881"
COMMENT     Sequence Label: U6_crRNA_mCherry
FEATURES             Location/Qualifiers
     source          1..6454
                     /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        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        544..923
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        924..1127
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1078..1098
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1172..1191
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1172..1190
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1231..1240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1237..1944
                     /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(1382..1400)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1650..1669
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1945..1971
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2045..2062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2051..2275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2321..2749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2408..2427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2618..2639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2758..2778)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2763..3092
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2943..3078
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3005..3024
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3159..3953
                     /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(3213..3232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3823..3842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4127..4248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4164..4183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4218..4237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4297..4313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4297..4313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4310..4332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4321..4337
                     /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(4345..4351,4352..4369,4370..4375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4390..4411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4528..4545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4699..5287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4779..4798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5458..6249,6250..6318))
                     /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     6081..6100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6319..6423)
                     /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 cttaattaag agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
      421 aaacaccgtt gatgtcacgg aacggagacg atatatcgtc tctttttttc aattgcatga
      481 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      541 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      601 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      661 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      721 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      781 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      841 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      901 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      961 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1021 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1081 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     1141 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     1201 ctggctagcg tttaaactta agcttccggt gccaccatgg tgagcaaggg cgaggaggat
     1261 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     1321 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     1381 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     1441 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     1501 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     1561 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     1621 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     1681 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     1741 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     1801 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     1861 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     1921 ggcggcatgg acgagctgta caagtaccca tacgatgttc cagattacgc ttaagaattc
     1981 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     2041 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2101 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2161 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2221 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2281 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2341 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2401 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2461 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2521 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2581 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2641 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2701 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2761 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2821 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2881 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2941 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3001 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3061 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3121 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3181 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3241 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3301 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3361 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3421 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3481 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3541 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3601 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3661 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3721 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3781 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3841 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3901 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3961 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4021 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4081 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4141 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4201 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4261 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4321 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4381 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4441 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4501 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4561 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4621 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4681 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4741 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4801 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4861 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4921 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4981 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5041 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5101 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5161 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5221 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5281 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5341 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5401 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5461 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5521 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5581 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5641 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5701 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5761 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5821 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5881 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5941 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6001 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6061 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6121 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6181 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6241 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6301 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6361 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6421 gcgcacattt ccccgaaaag tgccacctga cgtc
//
