LOCUS       sequence-208553-        7129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid expressing AsCpf1 under control of miniCMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7129)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 7129)
  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"
COMMENT     Sequence Label: pX551-miniCMV-AsCpf1
FEATURES             Location/Qualifiers
     source          1..7129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     146..165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        146..164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        183..221
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     218..242
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             274..4194
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
                     HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
                     AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
                     KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
                     ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
                     NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
                     RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
                     KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
                     KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
                     RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK
                     ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
                     LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
                     LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
                     HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
                     DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
                     HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR
                     SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
                     VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
                     FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
                     VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
                     HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
                     RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
                     KDLKLQNGISNQDWLAYIQELRN"
     regulatory      3914..3923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4195..4242
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4249..4329
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    4339..4387
                     /note="synthetic polyadenylation signal"
     repeat_region   4395..4535
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4395..4524
                     /label=AAV2 ITR
     rep_origin      4610..5065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4697..4716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4907..4928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5082..5101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5201..5223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5261..5279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5347..5451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5452..5520,5521..6312)
                     /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     complement(5670..5689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6483..7071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6972..6991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt cctgcggcct ctagataata cgactcacta tagggggatc cacgtatgtc
      181 gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga
      241 tcgcctggag gtaccgccac caccggtgcc accatgacac agttcgaggg ctttaccaac
      301 ctgtatcagg tgagcaagac actgcggttt gagctgatcc cacagggcaa gaccctgaag
      361 cacatccagg agcagggctt catcgaggag gacaaggccc gcaatgatca ctacaaggag
      421 ctgaagccca tcatcgatcg gatctacaag acctatgccg accagtgcct gcagctggtg
      481 cagctggatt gggagaacct gagcgccgcc atcgactcct atagaaagga gaaaaccgag
      541 gagacaagga acgccctgat cgaggagcag gccacatatc gcaatgccat ccacgactac
      601 ttcatcggcc ggacagacaa cctgaccgat gccatcaata agagacacgc cgagatctac
      661 aagggcctgt tcaaggccga gctgtttaat ggcaaggtgc tgaagcagct gggcaccgtg
      721 accacaaccg agcacgagaa cgccctgctg cggagcttcg acaagtttac aacctacttc
      781 tccggctttt atgagaacag gaagaacgtg ttcagcgccg aggatatcag cacagccatc
      841 ccacaccgca tcgtgcagga caacttcccc aagtttaagg agaattgtca catcttcaca
      901 cgcctgatca ccgccgtgcc cagcctgcgg gagcactttg agaacgtgaa gaaggccatc
      961 ggcatcttcg tgagcacctc catcgaggag gtgttttcct tcccttttta taaccagctg
     1021 ctgacacaga cccagatcga cctgtataac cagctgctgg gaggaatctc tcgggaggca
     1081 ggcaccgaga agatcaaggg cctgaacgag gtgctgaatc tggccatcca gaagaatgat
     1141 gagacagccc acatcatcgc ctccctgcca cacagattca tccccctgtt taagcagatc
     1201 ctgtccgata ggaacaccct gtctttcatc ctggaggagt ttaagagcga cgaggaagtg
     1261 atccagtcct tctgcaagta caagacactg ctgagaaacg agaacgtgct ggagacagcc
     1321 gaggccctgt ttaacgagct gaacagcatc gacctgacac acatcttcat cagccacaag
     1381 aagctggaga caatcagcag cgccctgtgc gaccactggg atacactgag gaatgccctg
     1441 tatgagcgga gaatctccga gctgacaggc aagatcacca agtctgccaa ggagaaggtg
     1501 cagcgcagcc tgaagcacga ggatatcaac ctgcaggaga tcatctctgc cgcaggcaag
     1561 gagctgagcg aggccttcaa gcagaaaacc agcgagatcc tgtcccacgc acacgccgcc
     1621 ctggatcagc cactgcctac aaccctgaag aagcaggagg agaaggagat cctgaagtct
     1681 cagctggaca gcctgctggg cctgtaccac ctgctggact ggtttgccgt ggatgagtcc
     1741 aacgaggtgg accccgagtt ctctgcccgg ctgaccggca tcaagctgga gatggagcct
     1801 tctctgagct tctacaacaa ggccagaaat tatgccacca agaagcccta ctccgtggag
     1861 aagttcaagc tgaactttca gatgcctaca ctggcctctg gctgggacgt gaataaggag
     1921 aagaacaatg gcgccatcct gtttgtgaag aacggcctgt actatctggg catcatgcca
     1981 aagcagaagg gcaggtataa ggccctgagc ttcgagccca cagagaaaac cagcgagggc
     2041 tttgataaga tgtactatga ctacttccct gatgccgcca agatgatccc aaagtgcagc
     2101 acccagctga aggccgtgac agcccacttt cagacccaca caacccccat cctgctgtcc
     2161 aacaatttca tcgagcctct ggagatcaca aaggagatct acgacctgaa caatcctgag
     2221 aaggagccaa agaagtttca gacagcctac gccaagaaaa ccggcgacca gaagggctac
     2281 agagaggccc tgtgcaagtg gatcgacttc acaagggatt ttctgtccaa gtataccaag
     2341 acaacctcta tcgatctgtc tagcctgcgg ccatcctctc agtataagga cctgggcgag
     2401 tactatgccg agctgaatcc cctgctgtac cacatcagct tccagagaat cgccgagaag
     2461 gagatcatgg atgccgtgga gacaggcaag ctgtacctgt tccagatcta taacaaggac
     2521 tttgccaagg gccaccacgg caagcctaat ctgcacacac tgtattggac cggcctgttt
     2581 tctccagaga acctggccaa gacaagcatc aagctgaatg gccaggccga gctgttctac
     2641 cgccctaagt ccaggatgaa gaggatggca caccggctgg gagagaagat gctgaacaag
     2701 aagctgaagg atcagaaaac cccaatcccc gacaccctgt accaggagct gtacgactat
     2761 gtgaatcaca gactgtccca cgacctgtct gatgaggcca gggccctgct gcccaacgtg
     2821 atcaccaagg aggtgtctca cgagatcatc aaggataggc gctttaccag cgacaagttc
     2881 tttttccacg tgcctatcac actgaactat caggccgcca attccccatc taagttcaac
     2941 cagagggtga atgcctacct gaaggagcac cccgagacac ctatcatcgg catcgatcgg
     3001 ggcgagagaa acctgatcta tatcacagtg atcgactcca ccggcaagat cctggagcag
     3061 cggagcctga acaccatcca gcagtttgat taccagaaga agctggacaa cagggagaag
     3121 gagagggtgg cagcaaggca ggcctggtct gtggtgggca caatcaagga tctgaagcag
     3181 ggctatctga gccaggtcat ccacgagatc gtggacctga tgatccacta ccaggccgtg
     3241 gtggtgctgg agaacctgaa tttcggcttt aagagcaaga ggaccggcat cgccgagaag
     3301 gccgtgtacc agcagttcga gaagatgctg atcgataagc tgaattgcct ggtgctgaag
     3361 gactatccag cagagaaagt gggaggcgtg ctgaacccat accagctgac agaccagttc
     3421 acctcctttg ccaagatggg cacccagtct ggcttcctgt tttacgtgcc tgccccatat
     3481 acatctaaga tcgatcccct gaccggcttc gtggacccct tcgtgtggaa aaccatcaag
     3541 aatcacgaga gccgcaagca cttcctggag ggcttcgact ttctgcacta cgacgtgaaa
     3601 accggcgact tcatcctgca ctttaagatg aacagaaatc tgtccttcca gaggggcctg
     3661 cccggcttta tgcctgcatg ggatatcgtg ttcgagaaga acgagacaca gtttgacgcc
     3721 aagggcaccc ctttcatcgc cggcaagaga atcgtgccag tgatcgagaa tcacagattc
     3781 accggcagat accgggacct gtatcctgcc aacgagctga tcgccctgct ggaggagaag
     3841 ggcatcgtgt tcagggatgg ctccaacatc ctgccaaagc tgctggagaa tgacgattct
     3901 cacgccatcg acaccatggt ggccctgatc cgcagcgtgc tgcagatgcg gaactccaat
     3961 gccgccacag gcgaggacta tatcaacagc cccgtgcgcg atctgaatgg cgtgtgcttc
     4021 gactcccggt ttcagaaccc agagtggccc atggacgccg atgccaatgg cgcctaccac
     4081 atcgccctga agggccagct gctgctgaat cacctgaagg agagcaagga tctgaagctg
     4141 cagaacggca tctccaatca ggactggctg gcctacatcc aggagctgcg caacaaaagg
     4201 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat
     4261 gttccagatt acgcttatcc ctacgacgtg cctgattatg catacccata tgatgtcccc
     4321 gactatgcct aagaattcaa taaaagatct ttattttcat tagatctgtg tgttggtttt
     4381 ttgtgtgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4441 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4501 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4561 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4621 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4681 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4741 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4801 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4861 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4921 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4981 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5041 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5101 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5161 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5221 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5281 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5341 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5401 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6001 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6061 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6121 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     6181 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6301 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6361 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6421 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6481 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6541 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6601 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6661 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6721 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6781 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6841 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6901 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6961 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7021 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7081 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcaca
//
