LOCUS       sequence-381222-        8415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic vector expressing CMV promoter driven AsCpf1, and U6 
            promoter driven guide RNA that target GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8415)
  AUTHORS   Haiting Ma, Rudolf Jaenisch
  TITLE     Multiplex genome editing of human pluripotent stem cells using Cpf1
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 8415)
  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"; journalName: "bioRxiv"
COMMENT     Sequence Label: CMV:AsCpf1-2A-GFP-U6-GFP-sg
FEATURES             Location/Qualifiers
     source          1..8415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          114..341
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      357..366
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             369..389
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             414..4331
                     /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      4051..4060
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4332..4379
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4407..4424)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4413..4620
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4629..4769
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4629..4758
                     /label=AAV2 ITR
     rep_origin      4844..5299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4931..4950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5141..5162
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5316..5335)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5435..5457
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5495..5513)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5581..5685
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5686..5754,5755..6546)
                     /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(5904..5923)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6717..7305
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7206..7225
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7367..7615
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7367..7387
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7538..7557
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     7637..7658
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_RNA        7666..7741
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        7838..8123
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
       61 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
      121 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
      181 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
      241 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
      301 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtggccgcca
      361 ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccacacagt
      421 tcgagggctt taccaacctg tatcaggtga gcaagacact gcggtttgag ctgatcccac
      481 agggcaagac cctgaagcac atccaggagc agggcttcat cgaggaggac aaggcccgca
      541 atgatcacta caaggagctg aagcccatca tcgatcggat ctacaagacc tatgccgacc
      601 agtgcctgca gctggtgcag ctggattggg agaacctgag cgccgccatc gactcctata
      661 gaaaggagaa aaccgaggag acaaggaacg ccctgatcga ggagcaggcc acatatcgca
      721 atgccatcca cgactacttc atcggccgga cagacaacct gaccgatgcc atcaataaga
      781 gacacgccga gatctacaag ggcctgttca aggccgagct gtttaatggc aaggtgctga
      841 agcagctggg caccgtgacc acaaccgagc acgagaacgc cctgctgcgg agcttcgaca
      901 agtttacaac ctacttctcc ggcttttatg agaacaggaa gaacgtgttc agcgccgagg
      961 atatcagcac agccatccca caccgcatcg tgcaggacaa cttccccaag tttaaggaga
     1021 attgtcacat cttcacacgc ctgatcaccg ccgtgcccag cctgcgggag cactttgaga
     1081 acgtgaagaa ggccatcggc atcttcgtga gcacctccat cgaggaggtg ttttccttcc
     1141 ctttttataa ccagctgctg acacagaccc agatcgacct gtataaccag ctgctgggag
     1201 gaatctctcg ggaggcaggc accgagaaga tcaagggcct gaacgaggtg ctgaatctgg
     1261 ccatccagaa gaatgatgag acagcccaca tcatcgcctc cctgccacac agattcatcc
     1321 ccctgtttaa gcagatcctg tccgatagga acaccctgtc tttcatcctg gaggagttta
     1381 agagcgacga ggaagtgatc cagtccttct gcaagtacaa gacactgctg agaaacgaga
     1441 acgtgctgga gacagccgag gccctgttta acgagctgaa cagcatcgac ctgacacaca
     1501 tcttcatcag ccacaagaag ctggagacaa tcagcagcgc cctgtgcgac cactgggata
     1561 cactgaggaa tgccctgtat gagcggagaa tctccgagct gacaggcaag atcaccaagt
     1621 ctgccaagga gaaggtgcag cgcagcctga agcacgagga tatcaacctg caggagatca
     1681 tctctgccgc aggcaaggag ctgagcgagg ccttcaagca gaaaaccagc gagatcctgt
     1741 cccacgcaca cgccgccctg gatcagccac tgcctacaac cctgaagaag caggaggaga
     1801 aggagatcct gaagtctcag ctggacagcc tgctgggcct gtaccacctg ctggactggt
     1861 ttgccgtgga tgagtccaac gaggtggacc ccgagttctc tgcccggctg accggcatca
     1921 agctggagat ggagccttct ctgagcttct acaacaaggc cagaaattat gccaccaaga
     1981 agccctactc cgtggagaag ttcaagctga actttcagat gcctacactg gcctctggct
     2041 gggacgtgaa taaggagaag aacaatggcg ccatcctgtt tgtgaagaac ggcctgtact
     2101 atctgggcat catgccaaag cagaagggca ggtataaggc cctgagcttc gagcccacag
     2161 agaaaaccag cgagggcttt gataagatgt actatgacta cttccctgat gccgccaaga
     2221 tgatcccaaa gtgcagcacc cagctgaagg ccgtgacagc ccactttcag acccacacaa
     2281 cccccatcct gctgtccaac aatttcatcg agcctctgga gatcacaaag gagatctacg
     2341 acctgaacaa tcctgagaag gagccaaaga agtttcagac agcctacgcc aagaaaaccg
     2401 gcgaccagaa gggctacaga gaggccctgt gcaagtggat cgacttcaca agggattttc
     2461 tgtccaagta taccaagaca acctctatcg atctgtctag cctgcggcca tcctctcagt
     2521 ataaggacct gggcgagtac tatgccgagc tgaatcccct gctgtaccac atcagcttcc
     2581 agagaatcgc cgagaaggag atcatggatg ccgtggagac aggcaagctg tacctgttcc
     2641 agatctataa caaggacttt gccaagggcc accacggcaa gcctaatctg cacacactgt
     2701 attggaccgg cctgttttct ccagagaacc tggccaagac aagcatcaag ctgaatggcc
     2761 aggccgagct gttctaccgc cctaagtcca ggatgaagag gatggcacac cggctgggag
     2821 agaagatgct gaacaagaag ctgaaggatc agaaaacccc aatccccgac accctgtacc
     2881 aggagctgta cgactatgtg aatcacagac tgtcccacga cctgtctgat gaggccaggg
     2941 ccctgctgcc caacgtgatc accaaggagg tgtctcacga gatcatcaag gataggcgct
     3001 ttaccagcga caagttcttt ttccacgtgc ctatcacact gaactatcag gccgccaatt
     3061 ccccatctaa gttcaaccag agggtgaatg cctacctgaa ggagcacccc gagacaccta
     3121 tcatcggcat cgatcggggc gagagaaacc tgatctatat cacagtgatc gactccaccg
     3181 gcaagatcct ggagcagcgg agcctgaaca ccatccagca gtttgattac cagaagaagc
     3241 tggacaacag ggagaaggag agggtggcag caaggcaggc ctggtctgtg gtgggcacaa
     3301 tcaaggatct gaagcagggc tatctgagcc aggtcatcca cgagatcgtg gacctgatga
     3361 tccactacca ggccgtggtg gtgctggaga acctgaattt cggctttaag agcaagagga
     3421 ccggcatcgc cgagaaggcc gtgtaccagc agttcgagaa gatgctgatc gataagctga
     3481 attgcctggt gctgaaggac tatccagcag agaaagtggg aggcgtgctg aacccatacc
     3541 agctgacaga ccagttcacc tcctttgcca agatgggcac ccagtctggc ttcctgtttt
     3601 acgtgcctgc cccatataca tctaagatcg atcccctgac cggcttcgtg gaccccttcg
     3661 tgtggaaaac catcaagaat cacgagagcc gcaagcactt cctggagggc ttcgactttc
     3721 tgcactacga cgtgaaaacc ggcgacttca tcctgcactt taagatgaac agaaatctgt
     3781 ccttccagag gggcctgccc ggctttatgc ctgcatggga tatcgtgttc gagaagaacg
     3841 agacacagtt tgacgccaag ggcacccctt tcatcgccgg caagagaatc gtgccagtga
     3901 tcgagaatca cagattcacc ggcagatacc gggacctgta tcctgccaac gagctgatcg
     3961 ccctgctgga ggagaagggc atcgtgttca gggatggctc caacatcctg ccaaagctgc
     4021 tggagaatga cgattctcac gccatcgaca ccatggtggc cctgatccgc agcgtgctgc
     4081 agatgcggaa ctccaatgcc gccacaggcg aggactatat caacagcccc gtgcgcgatc
     4141 tgaatggcgt gtgcttcgac tcccggtttc agaacccaga gtggcccatg gacgccgatg
     4201 ccaatggcgc ctaccacatc gccctgaagg gccagctgct gctgaatcac ctgaaggaga
     4261 gcaaggatct gaagctgcag aacggcatct ccaatcagga ctggctggcc tacatccagg
     4321 agctgcgcaa caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     4381 aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     4441 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4501 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4561 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     4621 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4681 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4741 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4801 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4861 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4921 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4981 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5041 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5101 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5161 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5221 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5281 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5341 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5401 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5461 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5521 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5581 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5641 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5701 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5761 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5821 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5881 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5941 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6001 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6061 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6121 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6181 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6241 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6301 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6361 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     6421 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6481 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6541 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6601 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6661 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6721 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6781 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6841 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6901 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6961 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7021 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7081 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7141 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7201 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7261 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7321 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc
     7381 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     7441 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     7501 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     7561 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtaat
     7621 ttctactctt gtagatcgtc gccgtccagc tcgaccaggt tttttgtttt agagctagaa
     7681 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     7741 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
     7801 tgcgccaatt ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg
     7861 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag
     7921 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     7981 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     8041 cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     8101 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     8161 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     8221 cgatgggggc gggggggggg ggggggncgc gccccgbggg gggggggggg gggggggggg
     8281 gggggggggg gggggggggg gggggggggg gggkgggggg ggggggkggg gggggggggg
     8341 gggggggggg gggggggggg gggggggggg gggngggggg gggggggggg ggggggggsg
     8401 gggggsgggg cgggg
//
