LOCUS       Exported                6898 bp ds-DNA     circular UNA 28-JUL-2022
DEFINITION  PKC1002.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jul 29, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6898
                     /organism="synthetic DNA construct"
                     /mol_type="genomic DNA"
     source          1286..3688
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     source          2903..3685
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     repeat_region   12..141
                     /label=AAV2 ITR
                     /note="color: #a6acb3"
     misc_feature    264..3688
                     /label=Geneious type: Editing History Insertion
                     /note="Geneious type: Editing History Insertion"
                     /note="/Original_Bases="
                     /note="color: #a6acb3"
     promoter        264..504
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff"
     promoter        505..512
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff"
     misc_feature    513..532
                     /label=HEKs3 spacer
                     /label=nonstandard type:  motif
                     /note="/Color=#ffff99"
                     /note="/created_by=jsfg"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3"
     misc_RNA        533..608
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(609..621)
                     /label=RT Template
                     /label=nonstandard type:  motif
                     /note="/Color=#ff7400"
                     /note="/created_by=jsfg"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3; direction: LEFT"
     variation       619..621
                     /label=CTT ins
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    complement(622..634)
                     /label=PBS
                     /label=nonstandard type:  motif
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    642..871
                     /label=H1 Promoter
                     /label=nonstandard type:  misc_marker
                     /note="/Color=#ff7400"
                     /note="/created_by=User"
                     /note="color: #a6acb3"
     gene            872..891
                     /label=PE3 HEKs3 ngRNA spacer
                     /note="/created_by=pkc"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_difference 892..974
                     /label=spCas9 Scaffold RNA
                     /note="/created_by=User"
                     /note="color: #a6acb3"
     promoter        1005..1255
                     /label=pEFS
                     /note="/created_by=User"
                     /note="color: #ffffff"
     regulatory      1286..1297
                     /label=KOZAK
                     /note="/created_by=jsfg"
                     /note="color: #ffffff"
     misc_feature    1295..1351
                     /label=bpNLS
                     /label=nonstandard type:  NLS
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     variation       1352..2839
                     /label=MMLV-RT Delta RH
                     /label=nonstandard type:  mutation
                     /note="/created_by=bretmiller"
                     /note="/modified_by=bretmiller"
                     /note="/modified_by=Joung Lab"
                     /note="color: #a6acb3"
     variation       1691..1693
                     /label=D114 (important in template binding)
                     /label=nonstandard type:  mutation
                     /note="/Color=#ffffbb"
                     /note="/created_by=bretmiller"
                     /note="/modified_by=bretmiller"
                     /note="color: #a6acb3"
     variation       1697..1699
                     /label=R116 (important for template binding)
                     /label=nonstandard type:  mutation
                     /note="/Color=#ffffbb"
                     /note="/created_by=bretmiller"
                     /note="color: #a6acb3"
     variation       1949..1951
                     /label=D200N
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     variation       2267..2269
                     /label=T306K
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     variation       2288..2290
                     /label=W313F
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     variation       2339..2341
                     /label=T330P
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    2840..2851
                     /label=linker
                     /label=nonstandard type:  linker
                     /note="/created_by=jsfg"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    2852..2902
                     /label=bpNLS
                     /label=nonstandard type:  NLS
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     CDS             2912..2968
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding 
                     separatepolypeptides."
                     /note="/Color=#0033aa"
                     /note="/modified_by=User"
                     /note="color: #993366"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2969..3685
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="/Color=#11ff11"
                     /note="/modified_by=User"
                     /note="color: #993366"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      3686..3688
                     /label=STOP
                     /note="/Color=#000000"
                     /note="/created_by=jsfg"
                     /note="color: #ffffff"
     misc_feature    3719..3724
                     /label=Geneious type: Editing History Insertion
                     /note="Geneious type: Editing History Insertion"
                     /note="/Original_Bases="
                     /note="color: #a6acb3"
     repeat_region   4161..4301
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #a6acb3"
     rep_origin      4376..4831
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5113..5217
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5218..6078
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /note="color: #ccffcc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5436..5455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      6249..6837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca gatgctgcac acttgtggac tactagtaag
      181 cttggatctt gaagaagctg caggaggtgc tggaggggaa gtggtccgga tccgatcagt
      241 gtgagggagt gtaaagctgg tttgagggcc tatttcccat gattccttca tatttgcata
      301 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      361 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      421 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      481 ctttatatat cttgtggaaa ggacgaaaca ccggcccaga ctgagcacgt gagttttaga
      541 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtgggaccga
      601 gtcggtcctc tgccatcaaa gcgtgctcag tctgtttttt tggaattcga acgctgacgt
      661 catcaacccg ctccaaggaa tcgcgggccc agtgtcacta ggcgggaaca cccagcgcgc
      721 gtgcgccctg gcaggaagat ggctgtgagg gacaggggag tggcgccctg caatatttgc
      781 atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc
      841 ttataagttc tgtatgagac cactttttcc cgtcaaccag tatcccggtg cgttttagag
      901 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      961 tcggtgcttt ttttctaact cgaacgctag ctgtgcgatc gttttcgagt ggctccggtg
     1021 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     1081 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     1141 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     1201 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggaccc
     1261 gacattagcg ctacagctta agcgggccgc caccatgaaa cggacagccg acggaagcga
     1321 gttcgagtca ccaaagaaga agcggaaagt caccctaaat atagaagatg agtatcggct
     1381 acatgagacc tcaaaagagc cagatgtttc tctagggtcc acatggctgt ctgattttcc
     1441 tcaggcctgg gcggaaaccg ggggcatggg actggcagtt cgccaagctc ctctgatcat
     1501 acctctgaaa gcaacctcta cccccgtgtc cataaaacaa taccccatgt cacaagaagc
     1561 cagactgggg atcaagcccc acatacagag actgttggac cagggaatac tggtaccctg
     1621 ccagtccccc tggaacacgc ccctgctacc cgttaagaaa ccagggacta atgattatag
     1681 gcctgtccag gatctgagag aagtcaacaa gcgggtggaa gacatccacc ccaccgtgcc
     1741 caacccttac aacctcttga gcgggctccc accgtcccac cagtggtaca ctgtgcttga
     1801 tttaaaggat gcctttttct gcctgagact ccaccccacc agtcagcctc tcttcgcctt
     1861 tgagtggaga gatccagaga tgggaatctc aggacaattg acctggacca gactcccaca
     1921 gggtttcaaa aacagtccca ccctgtttaa tgaggcactg cacagagacc tagcagactt
     1981 ccggatccag cacccagact tgatcctgct acagtacgtg gatgacttac tgctggccgc
     2041 cacttctgag ctagactgcc aacaaggtac tcgggccctg ttacaaaccc tagggaacct
     2101 cgggtatcgg gcctcggcca agaaagccca aatttgccag aaacaggtca agtatctggg
     2161 gtatcttcta aaagagggtc agagatggct gactgaggcc agaaaagaga ctgtgatggg
     2221 gcagcctact ccgaagaccc ctcgacaact aagggagttc ctagggaagg caggcttctg
     2281 tcgcctcttc atccctgggt ttgcagaaat ggcagccccc ctgtaccctc tcaccaaacc
     2341 ggggactctg tttaattggg gcccagacca acaaaaggcc tatcaagaaa tcaagcaagc
     2401 tcttctaact gccccagccc tggggttgcc agatttgact aagccctttg aactctttgt
     2461 cgacgagaag cagggctacg ccaaaggtgt cctaacgcaa aaactgggac cttggcgtcg
     2521 gccggtggcc tacctgtcca aaaagctaga cccagtagca gctgggtggc ccccttgcct
     2581 acggatggta gcagccattg ccgtactgac aaaggatgca ggcaagctaa ccatgggaca
     2641 gccactagtc attctggccc cccatgcagt agaggcacta gtcaaacaac cccccgaccg
     2701 ctggctttcc aacgcccgga tgactcacta tcaggccttg cttttggaca cggaccgggt
     2761 ccagttcgga ccggtggtag ccctgaaccc ggctacgctg ctcccactgc ctgaggaagg
     2821 gctgcaacac aactgccttt ctggcggctc aaaaagaacc gccgacggca gcgaattcga
     2881 gcccaagaag aagaggaaag tcggaagcgg agctactaac ttcagcctgc tgaagcaggc
     2941 tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagc tgttcaccgg
     3001 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3061 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3121 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctatg gagtgcagtg
     3181 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3241 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3301 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3361 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3421 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3481 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3541 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3601 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3661 tctcggcatg gacgagctgt acaagtaaaa acgttgttgt ttggggttga attactcttg
     3721 atcagataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3781 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3841 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttagttc ttgccacggc
     3901 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3961 caattccgtg gtgtttattt gtgaaatttg tgatgctatt gctttatttg taaccatcta
     4021 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4081 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     4141 aggtttttta aagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4201 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4261 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4321 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4381 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4441 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4501 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4561 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4621 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4681 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     4741 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4801 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4861 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4921 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4981 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5041 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5221 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5281 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5341 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5401 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5461 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5521 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5581 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5641 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5701 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5761 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5821 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5881 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5941 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6001 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6061 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6121 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6181 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6241 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6301 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6361 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     6421 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6481 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6541 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6601 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6661 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6721 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6781 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6841 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
