LOCUS       Exported                8470 bp DNA     circular SYN 07-JUN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 27, 2022 from SnapGene Viewer 6.0.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        83..1678
                     /label=CAG promoter
     promoter        390..665
                     /label=chicken beta-actin promoter
     promoter        1739..1755
                     /label=T7 promoter
     misc_feature    1757..1765
                     /label=Kozak sequence
     CDS             1769..1789
                     /codon_start=1
                     /label=NLS
                     /translation="PKKKRKV"
     CDS             1796..4957
                     /codon_start=1
                     /label=Cas12i2
                     /translation="MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFG
                     GITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTAS
                     DAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQ
                     NNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETF
                     RQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVE
                     QNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFF
                     DSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRN
                     ILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQR
                     NDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFG
                     YHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRI
                     PVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDI
                     VSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFD
                     AICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLST
                     LETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVE
                     RIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHN
                     ITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGE
                     YYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRG
                     GRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRI
                     KLQLTS"
     CDS             4964..4984
                     /codon_start=1
                     /label=NLS
                     /translation="PKKKRKV"
     CDS             4991..5080
                     /codon_start=1
                     /label=3xHA tag
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5102..5155
                     /codon_start=1
                     /label=T2A peptide
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5156..5872
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5918..6039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6082..6098)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6106..6122
                     /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(6130..6160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     rep_origin      complement(6484..7072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7243..8103)
                     /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"
     promoter        complement(8104..8208)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
     1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagcgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcta gtgaattcta
     1741 atacgactca ctataggccg ccaccatgcc caagaagaag aggaaggttc ccgggatgag
     1801 cagcgccatc aagagctaca agagcgtgct gcgccccaac gagcgcaaga accagctgct
     1861 gaagagcacc atccagtgcc tggaggacgg cagcgccttc ttcttcaaga tgctgcaggg
     1921 cctgttcggc ggcatcaccc ccgagatcgt gcgcttcagc accgagcagg agaagcagca
     1981 gcaggacatc gccctgtggt gcgccgtgaa ctggttccgc cccgtgagcc aggacagcct
     2041 gacccacacc atcgccagcg acaacctggt ggagaagttc gaggagtact acggcggcac
     2101 cgccagcgac gccatcaagc agtacttcag cgccagcatc ggcgagagct actactggaa
     2161 cgactgccgc cagcagtact acgacctgtg ccgcgagctg ggcgtggagg tgagcgacct
     2221 gacccacgac ctggagatcc tgtgccgcga gaagtgcctg gccgtggcca ccgagagcaa
     2281 ccagaacaac agcatcatca gcgtgctgtt cggcaccggc gagaaggagg accgcagcgt
     2341 gaagctgcgc atcaccaaga agatcctgga ggccatcagc aacctgaagg agatccccaa
     2401 gaacgtggcc cccatccagg agatcatcct gaacgtggcc aaggccacca aggagacctt
     2461 ccgccaggtg tacgccggca acctgggcgc ccccagcacc ctggagaagt tcatcgccaa
     2521 ggacggccag aaggagttcg acctgaagaa gctgcagacc gacctgaaga aggtgatccg
     2581 cggcaagagc aaggagcgcg actggtgctg ccaggaggag ctgcgcagct acgtggagca
     2641 gaacaccatc cagtacgacc tgtgggcctg gggcgagatg ttcaacaagg cccacaccgc
     2701 cctgaagatc aagagcaccc gcaactacaa cttcgccaag cagcgcctgg agcagttcaa
     2761 ggagatccag agcctgaaca acctgctggt ggtgaagaag ctgaacgact tcttcgacag
     2821 cgagttcttc agcggcgagg agacctacac catctgcgtg caccacctgg gcggcaagga
     2881 cctgagcaag ctgtacaagg cctgggagga cgaccccgcc gaccccgaga acgccatcgt
     2941 ggtgctgtgc gacgacctga agaacaactt caagaaggag cccatccgca acatcctgcg
     3001 ctacatcttc accatccgcc aggagtgcag cgcccaggac atcctggccg ccgccaagta
     3061 caaccagcag ctggaccgct acaagagcca gaaggccaac cccagcgtgc tgggcaacca
     3121 gggcttcacc tggaccaacg ccgtgatcct gcccgagaag gcccagcgca acgaccgccc
     3181 caacagcctg gacctgcgca tctggctgta cctgaagctg cgccaccccg acggccgctg
     3241 gaagaagcac cacatcccct tctacgacac ccgcttcttc caggagatct acgccgccgg
     3301 caacagcccc gtggacacct gccagttccg caccccccgc ttcggctacc acctgcccaa
     3361 gctgaccgac cagaccgcca tccgcgtgaa caagaagcac gtgaaggccg ccaagaccga
     3421 ggcccgcatc cgcctggcca tccagcaggg caccctgccc gtgagcaacc tgaagatcac
     3481 cgagatcagc gccaccatca acagcaaggg ccaggtgcgc atccccgtga agttcgacgt
     3541 gggccgccag aagggcaccc tgcagatcgg cgaccgcttc tgcggctacg accagaacca
     3601 gaccgccagc cacgcctaca gcctgtggga ggtggtgaag gagggccagt accacaagga
     3661 gctgggctgc ttcgtgcgct tcatcagcag cggcgacatc gtgagcatca ccgagaaccg
     3721 cggcaaccag ttcgaccagc tgagctacga gggcctggcc tacccccagt acgccgactg
     3781 gcgcaagaag gccagcaagt tcgtgagcct gtggcagatc accaagaaga acaagaagaa
     3841 ggagatcgtg accgtggagg ccaaggagaa gttcgacgcc atctgcaagt accagccccg
     3901 cctgtacaag ttcaacaagg agtacgccta cctgctgcgc gacatcgtgc gcggcaagag
     3961 cctggtggag ctgcagcaga tccgccagga gatcttccgc ttcatcgagc aggactgcgg
     4021 cgtgacccgc ctgggcagcc tgagcctgag caccctggag accgtgaagg ccgtgaaggg
     4081 catcatctac agctacttca gcaccgccct gaacgccagc aagaacaacc ccatcagcga
     4141 cgagcagcgc aaggagttcg accccgagct gttcgccctg ctggagaagc tggagctgat
     4201 ccgcacccgc aagaagaagc agaaggtgga gcgcatcgcc aacagcctga tccagacctg
     4261 cctggagaac aacatcaagt tcatccgcgg cgagggcgac ctgagcacca ccaacaacgc
     4321 caccaagaag aaggccaaca gccgcagcat ggactggctg gcccgcggcg tgttcaacaa
     4381 gatccgccag ctggccccca tgcacaacat caccctgttc ggctgcggca gcctgtacac
     4441 cagccaccag gaccccctgg tgcaccgcaa ccccgacaag gccatgaagt gccgctgggc
     4501 cgccatcccc gtgaaggaca tcggcgactg ggtgctgcgc aagctgagcc agaacctgcg
     4561 cgccaagaac atcggcaccg gcgagtacta ccaccagggc gtgaaggagt tcctgagcca
     4621 ctacgagctg caggacctgg aggaggagct gctgaagtgg cgcagcgacc gcaagagcaa
     4681 catcccctgc tgggtgctgc agaaccgcct ggccgagaag ctgggcaaca aggaggccgt
     4741 ggtgtacatc cccgtgcgcg gcggccgcat ctacttcgcc acccacaagg tggccaccgg
     4801 cgccgtgagc atcgtgttcg accagaagca ggtgtgggtg tgcaacgccg accacgtggc
     4861 cgccgccaac atcgccctga ccgtgaaggg catcggcgag cagagcagcg acgaggagaa
     4921 ccccgacggc agccgcatca agctgcagct gaccagcgct agcccaaaga agaagaggaa
     4981 agtctctaga tacccttatg atgttccaga ttatgccgga tacccatacg atgtccctga
     5041 ctatgcaggc tcctaccctt atgacgtccc agactacgcc ggatccaggt ccggcggcgg
     5101 agagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatggt
     5161 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     5221 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     5281 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     5341 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     5401 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     5461 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     5521 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     5581 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     5641 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     5701 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     5761 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     5821 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgca
     5881 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     5941 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     6001 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat taattaacgt cgacctctag
     6061 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6121 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt
     6181 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6241 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6301 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6361 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6421 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6481 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6541 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6601 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6661 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6721 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6781 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6841 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6901 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6961 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7021 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7081 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7141 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7201 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7261 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7321 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7381 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7441 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7501 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7561 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7621 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7681 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7741 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7801 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7861 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7921 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7981 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8041 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8101 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8161 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8221 aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac
     8281 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     8341 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     8401 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     8461 ggccagatat
//
