LOCUS       sequence-394522-        7961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Csx29 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7961)
  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 7961)
  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"; doi: 
            "10.1126/science.add7450"; journalName: "Science"; date: 
            "2022-11-25- 25"; volume: "378"; issue: "6622"; pages: "874-881"
COMMENT     Sequence Label: pCDNA3_Csx29
FEATURES             Location/Qualifiers
     source          1..7961
                     /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        538..917
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        918..1121
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1072..1092
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1166..1185
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1166..1184
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1754..1763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3552..3569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3558..3782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3828..4256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3915..3934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4125..4146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4265..4285)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4270..4599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4450..4585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4512..4531
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4666..5460
                     /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(4720..4739)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5330..5349
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5634..5755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5671..5690)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5725..5744
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5804..5820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5804..5820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5817..5839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5828..5844
                     /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(5852..5858,5859..5876,5877..5882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5897..5918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6035..6052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6206..6794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6286..6305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6965..7756,7757..7825))
                     /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     7588..7607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7826..7930)
                     /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 gaaacaccgt
      421 aatttctact cttgtagatg gagacgatat atcgtctctt tttcaattgc atgaagaatc
      481 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      541 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      601 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      661 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      721 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      781 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      841 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      901 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      961 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     1021 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     1081 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     1141 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     1201 agcgtttaaa cttaagcttg ccaccatgag caatcctatc agagatatcc aggacagact
     1261 gaagaccgcc aagttcgaca acaaggacga catgatgaac ctggcttcaa gcctgtacaa
     1321 gtacgagaaa cagctgatgg acagctctga ggctacactg tgccagcagg gcctgagcaa
     1381 ccggcctaac tctttttctc agctgagcca atttcgggac agcgacattc agagcaaggc
     1441 cggcggccag accggtaagt tctggcagaa cgagtacgag gcttgtaaaa acttccagac
     1501 acacaaggag agaagagaaa ccctggaaca gatcatcaga ttcctgcaaa atggcgctga
     1561 ggaaaaggac gctgatgatc tgctgctgaa gaccctggcc cgagcctact tccaccgtgg
     1621 actgctgtat agacctaagg gcttcagcgt gcctgcgagg aaggtggaag ccatgaaaaa
     1681 ggccatcgcc tactgcgaaa tcatccttga taagaatgag gaagaatcgg aggctctgag
     1741 aatctggctg tacgccgcca tggaactccg gagatgcggc gaggagtatc ctgagaactt
     1801 tgcggagaag ctgttctacc ttgccaacga cggcttcatc agcgagctgt acgacatccg
     1861 gctgttcctg gaatacaccg agagagaaga ggacaacaac ttcctggaca tgatcctgca
     1921 ggagaatcag gacagagaaa gactgttcga gctgtgtctg tacaaggcca gagcctgttt
     1981 tcacctgaac cagctgaacg acgtgcggat ctacggcgag agcgcaatcg acaacgcccc
     2041 tggcgccttc gccgatcctt tctgggatga actggtggag ttcatcagaa tgctgcggaa
     2101 caagaagtct gaactgtgga aggaaattgc catcaaggcc tgggacaagt gcagagaaaa
     2161 ggaaatgaag gtgggcaaca acatctacct ttcttggtac tgggccagac agagagagct
     2221 gtacgacctg gccttcatgg cccaagacgg catcgagaag aaaacaagaa tcgccgatag
     2281 cctgaagagc cggaccaccc tgagaatcca ggagctgaat gagctgagaa aggatgccca
     2341 ccggaagcag aaccgtcggc tggaagataa gctggatcgg atcatcgagc aggagaacga
     2401 ggccagagac ggcgcctacc tgcggagaaa ccccccctgc ttcaccggcg gaaaaagaga
     2461 ggaaatcccc tttgccagac ttccacagaa ctggatcgcc gtgcacttct atctgaacga
     2521 gctggaaagc cacgaaggcg gcaagggcgg ccacgccctg atctacgacc ctcagaaggc
     2581 tgagaaagat cagtggcaag acaagagctt cgactacaaa gaactgcaca gaaaattcct
     2641 cgagtggcag gagaactaca tcctgaacga ggagggcagc gccgatttcc tggtgacact
     2701 gtgcagagag atcgagaaag ccatgccttt cctgtttaag tccgaggtga tccccgagga
     2761 cagacccgtg ctgtggattc ctcacggctt cctgcataga ctgcctctgc acgccgctat
     2821 gaagagcgga aacaattcta atatcgagat cttctgggag cggcacgcca gcagatatct
     2881 ccctgcttgg cacctgttcg atcctgcccc atacagccgc gaggaaagct ccaccctgct
     2941 gaagaacttc gaggagtacg acttccagaa cctggaaaac ggcgaaatcg aggtctacgc
     3001 ccccagcagc cccaagaagg tgaaggaagc catccgggaa aaccccgcta ttctgctgct
     3061 gctgtgtcac ggcgaggccg acatgacaaa ccctttccgg agctgcctga agctgaaaaa
     3121 caaagatatg accatcttcg acctgctgac cgtggaagat gtgaggctgt ctggcagcag
     3181 aatccttctg ggagcctgcg agtctgacat ggtgccacct ctggagttca gcgtggacga
     3241 acacctgagc gtgtccggcg cttttctcag ccataaggcc ggagaaatcg tcgccggcct
     3301 gtggaccgtg gactctgaga aggttgacga gtgctacagc tacctggtgg aagagaagga
     3361 cttcctgaga aacctgcagg agtggcagat ggccgagaca gagaacttca gaagcgagaa
     3421 tgatagctcc ctgttctaca agatcgcccc atttcggatc attggctttc ctgccgaatg
     3481 agaattctgc agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac
     3541 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3601 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3661 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3721 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3781 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3841 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3901 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3961 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4021 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4081 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4141 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4201 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     4261 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4321 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4381 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4441 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4501 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4561 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4621 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4681 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4741 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4801 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4861 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4921 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4981 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5041 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5101 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5161 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     5221 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5281 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5341 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5401 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5461 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5521 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5581 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5641 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5701 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5761 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5821 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5881 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5941 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6001 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     6061 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     7021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     7081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7921 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
