LOCUS       sequence-431470-        6524 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Fragmid fragment: (Cas protein) unmodified AsCas12a; not preferred.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6524)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 6524)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA181
FEATURES             Location/Qualifiers
     source          1..6524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             445..4362
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      4082..4091
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4434..4450)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4434..4450)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4447..4469)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4458..4474
                     /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(4482..4488,4489..4506,4507..4512))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4527..4548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4665..4682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4836..5424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4916..4935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5602..6411)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6325..6344
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6403..6422
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagactgg cagcacacag ttcgagggct ttaccaacct gtatcaggtg
      481 agcaagacac tgcggtttga gctgatccca cagggcaaga ccctgaagca catccaggag
      541 cagggcttca tcgaggagga caaggcccgc aatgatcact acaaggagct gaagcccatc
      601 atcgatcgga tctacaagac ctatgccgac cagtgcctgc agctggtgca gctggattgg
      661 gagaacctga gcgccgccat cgactcctat agaaaggaga aaaccgagga gacaaggaac
      721 gccctgatcg aggagcaggc cacatatcgc aatgccatcc acgactactt catcggccgg
      781 acagacaacc tgaccgatgc catcaataag agacacgccg agatctacaa gggcctgttc
      841 aaggccgagc tgtttaatgg caaggtgctg aagcagctgg gcaccgtgac cacaaccgag
      901 cacgagaacg ccctgctgcg gagcttcgac aagtttacaa cctacttctc cggcttttat
      961 gagaacagga agaacgtgtt cagcgccgag gatatcagca cagccatccc acaccgcatc
     1021 gtgcaggaca acttccccaa gtttaaggag aattgtcaca tcttcacacg cctgatcacc
     1081 gccgtgccca gcctgcggga gcactttgag aacgtgaaga aggccatcgg catcttcgtg
     1141 agcacctcca tcgaggaggt gttttccttc cctttttata accagctgct gacacagacc
     1201 cagatcgacc tgtataacca gctgctggga ggaatctctc gggaggcagg caccgagaag
     1261 atcaagggcc tgaacgaggt gctgaatctg gccatccaga agaatgatga gacagcccac
     1321 atcatcgcct ccctgccaca cagattcatc cccctgttta agcagatcct gtccgatagg
     1381 aacaccctgt ctttcatcct ggaggagttt aagagcgacg aggaagtgat ccagtccttc
     1441 tgcaagtaca agacactgct gagaaacgag aacgtgctgg agacagccga ggccctgttt
     1501 aacgagctga acagcatcga cctgacacac atcttcatca gccacaagaa gctggagaca
     1561 atcagcagcg ccctgtgcga ccactgggat acactgagga atgccctgta tgagcggaga
     1621 atctccgagc tgacaggcaa gatcaccaag tctgccaagg agaaggtgca gcgcagcctg
     1681 aagcacgagg atatcaacct gcaggagatc atctctgccg caggcaagga gctgagcgag
     1741 gccttcaagc agaaaaccag cgagatcctg tcccacgcac acgccgccct ggatcagcca
     1801 ctgcctacaa ccctgaagaa gcaggaggag aaggagatcc tgaagtctca gctggacagc
     1861 ctgctgggcc tgtaccacct gctggactgg tttgccgtgg atgagtccaa cgaggtggac
     1921 cccgagttct ctgcccggct gaccggcatc aagctggaga tggagccttc tctgagcttc
     1981 tacaacaagg ccagaaatta tgccaccaag aagccctact ccgtggagaa gttcaagctg
     2041 aactttcaga tgcctacact ggcctctggc tgggacgtga ataaggagaa gaacaatggc
     2101 gccatcctgt ttgtgaagaa cggcctgtac tatctgggca tcatgccaaa gcagaagggc
     2161 aggtataagg ccctgagctt cgagcccaca gagaaaacca gcgagggctt tgataagatg
     2221 tactatgact acttccctga tgccgccaag atgatcccaa agtgcagcac ccagctgaag
     2281 gccgtgacag cccactttca gacccacaca acccccatcc tgctgtccaa caatttcatc
     2341 gagcctctgg agatcacaaa ggagatctac gacctgaaca atcctgagaa ggagccaaag
     2401 aagtttcaga cagcctacgc caagaaaacc ggcgaccaga agggctacag agaggccctg
     2461 tgcaagtgga tcgacttcac aagggatttt ctgtccaagt ataccaagac aacctctatc
     2521 gatctgtcta gcctgcggcc atcctctcag tataaggacc tgggcgagta ctatgccgag
     2581 ctgaatcccc tgctgtacca catcagcttc cagagaatcg ccgagaagga gatcatggat
     2641 gccgtggaga caggcaagct gtacctgttc cagatctata acaaggactt tgccaagggc
     2701 caccacggca agcctaatct gcacacactg tattggaccg gcctgttttc tccagagaac
     2761 ctggccaaga caagcatcaa gctgaatggc caggccgagc tgttctaccg ccctaagtcc
     2821 aggatgaaga ggatggcaca ccggctggga gagaagatgc tgaacaagaa gctgaaggat
     2881 cagaaaaccc caatccccga caccctgtac caggagctgt acgactatgt gaatcacaga
     2941 ctgtcccacg acctgtctga tgaggccagg gccctgctgc ccaacgtgat caccaaggag
     3001 gtgtctcacg agatcatcaa ggataggcgc tttaccagcg acaagttctt tttccacgtg
     3061 cctatcacac tgaactatca ggccgccaat tccccatcta agttcaacca gagggtgaat
     3121 gcctacctga aggagcaccc cgagacacct atcatcggca tcgatcgggg cgagagaaac
     3181 ctgatctata tcacagtgat cgactccacc ggcaagatcc tggagcagcg gagcctgaac
     3241 accatccagc agtttgatta ccagaagaag ctggacaaca gggagaagga gagggtggca
     3301 gcaaggcagg cctggtctgt ggtgggcaca atcaaggatc tgaagcaggg ctatctgagc
     3361 caggtcatcc acgagatcgt ggacctgatg atccactacc aggccgtggt ggtgctggag
     3421 aacctgaatt tcggctttaa gagcaagagg accggcatcg ccgagaaggc cgtgtaccag
     3481 cagttcgaga agatgctgat cgataagctg aattgcctgg tgctgaagga ctatccagca
     3541 gagaaagtgg gaggcgtgct gaacccatac cagctgacag accagttcac ctcctttgcc
     3601 aagatgggca cccagtctgg cttcctgttt tacgtgcctg ccccatatac atctaagatc
     3661 gatcccctga ccggcttcgt ggaccccttc gtgtggaaaa ccatcaagaa tcacgagagc
     3721 cgcaagcact tcctggaggg cttcgacttt ctgcactacg acgtgaaaac cggcgacttc
     3781 atcctgcact ttaagatgaa cagaaatctg tccttccaga ggggcctgcc cggctttatg
     3841 cctgcatggg atatcgtgtt cgagaagaac gagacacagt ttgacgccaa gggcacccct
     3901 ttcatcgccg gcaagagaat cgtgccagtg atcgagaatc acagattcac cggcagatac
     3961 cgggacctgt atcctgccaa cgagctgatc gccctgctgg aggagaaggg catcgtgttc
     4021 agggatggct ccaacatcct gccaaagctg ctggagaatg acgattctca cgccatcgac
     4081 accatggtgg ccctgatccg cagcgtgctg cagatgcgga actccaatgc cgccacaggc
     4141 gaggactata tcaacagccc cgtgcgcgat ctgaatggcg tgtgcttcga ctcccggttt
     4201 cagaacccag agtggcccat ggacgccgat gccaatggcg cctaccacat cgccctgaag
     4261 ggccagctgc tgctgaatca cctgaaggag agcaaggatc tgaagctgca gaacggcatc
     4321 tccaatcagg actggctggc ctacatccag gagctgcgca acgggtccaa gtcttcatcg
     4381 gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggtgtaatc atggtcatag
     4441 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4501 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4561 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4621 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4681 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4741 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4801 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4861 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4921 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4981 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5041 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5101 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5161 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5221 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5281 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5341 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5401 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5461 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5521 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa gcccaatctg aataatgtta
     5581 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     5641 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     5701 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     5761 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     5821 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     5881 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     5941 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     6001 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     6061 attttcacct gaatcaggat attcttctaa tacctggaat gctgtttttc cggggatcgc
     6121 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     6181 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     6241 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca agcgatagat
     6301 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     6361 catgttggaa tttaatcgcg gcctcgacgt ttcccgttga atatggctca taacacccct
     6421 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg
     6481 tgcaatgtaa catcagagat tttgagacac gggccagagc tgca
//
