LOCUS       Exported                7521 bp ds-DNA     circular SYN 04-MAR-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7521)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Mar 9, 2022 from SnapGene 4.3.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7521
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          142..147
                     /label=Source_1
                     /note="color: #ffffff"
     source          4563..4567
                     /label=Source_1
                     /note="color: #ffffff"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     misc_feature    142..147
                     /label=Misc_Feature_3
                     /note="XbaI"
                     /note="color: #a6acb3"
     promoter        148..404
                     /label=U1A
                     /note="color: #ffffff; direction: RIGHT"
     CDS             411..518
                     /codon_start=1
                     /label=c-intein
                     /note="color: #000000"
                     /translation="MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN"
     CDS             519..2903
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /note="This feature has 3 segments:
                        1:  519 .. 1316 / #993366
                        2: 1317 .. 1319 / #cc99b2
                        3: 1320 .. 2903 / #993366"
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             2904..3002
                     /codon_start=1
                     /label=Linker
                     /note="color: #a6acb3"
                     /translation="SGGSSGGSSGSETPGTSESATPESSGGSSGGSS"
     CDS             3003..4493
                     /codon_start=1
                     /label=mlv
                     /note="color: #ff00ff"
                     /translation="TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQ
                     APLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKP
                     GTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
                     SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQY
                     VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT
                     EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQ
                     QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL
                     DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMT
                     HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD"
     misc_feature    4494..4535
                     /label=SV40
                     /note="color: #a6acb3"
     CDS             4536..4556
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     polyA_signal    4568..4775
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   4784..4924
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     rep_origin      4999..5454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5736..5840
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5841..6701
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5841 .. 5909 / #ccffcc / signal sequence
                        2: 5910 .. 6701 / #ccffcc
                     Cleavage site after base 5909"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6872..7460
                     /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 ttctagaatg gaggcggtac tatgtagatg agaattcagg
      181 agcaaactgg gaaaagcaac tgcttccaaa tatttgtgat ttttacagtg tagttttgga
      241 aaaactctta gcctaccaat tcttctaagt gttttaaaat gtgggagcca gtacacatga
      301 agttatagag tgttttaatg aggcttaaat atttaccgta actatgaaat gctacgcata
      361 tcatgctgtt caggctccgt ggccacgcaa ctcatactac cggtgccacc atgatcaaga
      421 ttgctacacg gaaatacctg ggaaagcaga acgtgtacga catcggcgtg gagcgggatc
      481 acaacttcgc cctgaagaat ggctttatcg ccagcaattg cttcgactcc gtggaaatct
      541 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
      601 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
      661 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
      721 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
      781 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
      841 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
      901 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
      961 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     1021 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     1081 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     1141 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     1201 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     1261 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggacgcta
     1321 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     1381 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     1441 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     1501 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     1561 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     1621 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     1681 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     1741 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     1801 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     1861 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     1921 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     1981 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     2041 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     2101 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     2161 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     2221 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     2281 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     2341 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     2401 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     2461 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     2521 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     2581 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     2641 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     2701 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     2761 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     2821 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     2881 acctgtctca gctgggaggt gactctggag gatctagcgg aggatcctct ggcagcgaga
     2941 caccaggaac aagcgagtca gcaacaccag agagcagtgg cggcagcagc ggcggcagca
     3001 gcaccctaaa tatagaagat gagtatcggc tacatgagac ctcaaaagag ccagatgttt
     3061 ctctagggtc cacatggctg tctgattttc ctcaggcctg ggcggaaacc gggggcatgg
     3121 gactggcagt tcgccaagct cctctgatca tacctctgaa agcaacctct acccccgtgt
     3181 ccataaaaca ataccccatg tcacaagaag ccagactggg gatcaagccc cacatacaga
     3241 gactgttgga ccagggaata ctggtaccct gccagtcccc ctggaacacg cccctgctac
     3301 ccgttaagaa accagggact aatgattata ggcctgtcca ggatctgaga gaagtcaaca
     3361 agcgggtgga agacatccac cccaccgtgc ccaaccctta caacctcttg agcgggctcc
     3421 caccgtccca ccagtggtac actgtgcttg atttaaagga tgcctttttc tgcctgagac
     3481 tccaccccac cagtcagcct ctcttcgcct ttgagtggag agatccagag atgggaatct
     3541 caggacaatt gacctggacc agactcccac agggtttcaa aaacagtccc accctgttta
     3601 atgaggcact gcacagagac ctagcagact tccggatcca gcacccagac ttgatcctgc
     3661 tacagtacgt ggatgactta ctgctggccg ccacttctga gctagactgc caacaaggta
     3721 ctcgggccct gttacaaacc ctagggaacc tcgggtatcg ggcctcggcc aagaaagccc
     3781 aaatttgcca gaaacaggtc aagtatctgg ggtatcttct aaaagagggt cagagatggc
     3841 tgactgaggc cagaaaagag actgtgatgg ggcagcctac tccgaagacc cctcgacaac
     3901 taagggagtt cctagggaag gcaggcttct gtcgcctctt catccctggg tttgcagaaa
     3961 tggcagcccc cctgtaccct ctcaccaaac cggggactct gtttaattgg ggcccagacc
     4021 aacaaaaggc ctatcaagaa atcaagcaag ctcttctaac tgccccagcc ctggggttgc
     4081 cagatttgac taagcccttt gaactctttg tcgacgagaa gcagggctac gccaaaggtg
     4141 tcctaacgca aaaactggga ccttggcgtc ggccggtggc ctacctgtcc aaaaagctag
     4201 acccagtagc agctgggtgg cccccttgcc tacggatggt agcagccatt gccgtactga
     4261 caaaggatgc aggcaagcta accatgggac agccactagt cattctggcc ccccatgcag
     4321 tagaggcact agtcaaacaa ccccccgacc gctggctttc caacgcccgg atgactcact
     4381 atcaggcctt gcttttggac acggaccggg tccagttcgg accggtggta gccctgaacc
     4441 cggctacgct gctcccactg cctgaggaag ggctgcaaca caactgcctt gattctggcg
     4501 gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaag
     4561 aaaattcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4621 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4681 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4741 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     4801 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4861 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4921 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4981 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5041 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5341 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5401 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5521 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5581 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5641 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5701 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5761 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5821 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5941 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6001 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6061 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6121 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6181 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6241 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6301 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6361 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6421 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6481 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6541 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6601 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6661 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6721 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6781 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6841 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6901 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6961 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     7021 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7081 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7141 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7201 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7261 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7321 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7381 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7441 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7501 gctggccttt tgctcacatg t
//
