LOCUS       Exported                7373 bp ds-DNA     circular SYN 29-JUN-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jan 2, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7373
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             412..465
                     /codon_start=1
                     /label=BPSV40 NLS
                     /translation="KRTADGSEFESPKKKRKV"
     CDS             466..3624
                     /codon_start=1
                     /label=SlugCas9-NNG
                     /translation="NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEG
                     RRSKRGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDEL
                     VIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQ
                     VRGEKNRFKTADIIKEIIQLLNVQKNFHQLDENFINKYIELVEMRREYFEGPGKGSPYG
                     WEGDPKAWYETLMGHCTYFPDELRSVKYAYSADLFNALNDLNNLVIQRDGLSKLEYHEK
                     YHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQ
                     SILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLK
                     CIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPVVKRTFGQA
                     INLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQN
                     AKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKVLV
                     KQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINK
                     FEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKE
                     RNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYSEMF
                     IIPKQVQDIKDFRNFKYSHRVDKKPNRRLINDTLYSTRKKDNSTYIVQTIKDIYAKDNT
                     TLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNN
                     GPIVKSLKYIGNKLGRHLDVTHQFKSSTKKLVKRSIKPYRFDVYLTDKGYKFITISYLD
                     VLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVSSDTRNM
                     IELDLPDIRYKEYCELNNIKGKPRIIKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLL
                     FKRGN"
     misc_feature    2806..2808
                     /label=Q782R
     misc_feature    3124..3125
                     /label=S888R
     misc_feature    3178..3180
                     /label=L906R
     misc_feature    3412..3414
                     /label=N984S
     misc_feature    3497..3498
                     /label=E1012K
     misc_feature    3508..3510
                     /label=K1016I
     CDS             3667..3687
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3697..3753
                     /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 separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3754..4350
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             4363..4383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4392..4409
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4438..4662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4733..4749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4757..4773
                     /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(4781..4811)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4826..4847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5131..5719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5890..6750)
                     /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(6751..6855)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7125..7373
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcaacca aaaattcata
      481 ctgggactgg acatcggaat caccagcgtg ggctacggcc tgatcgacta cgagacaaag
      541 aatatcatcg atgccggcgt tagactgttc cccgaggcca acgtggaaaa caacgaggga
      601 agaaggtcca aacgtggaag cagaagactg aagcgacgcc gcattcacag acttgaacgg
      661 gtgaagaagc tgctcgagga ttataatctg ctggatcagt cccagattcc tcagtctaca
      721 aacccctacg ccatccgcgt gaagggcctg tctgaagccc tgagcaagga cgaactcgtg
      781 attgccctgc tccatatcgc caagagaaga ggcatccaca agatcgacgt gatcgacagc
      841 aacgacgacg tggggaacga gctcagcacc aaggaacagc tgaataagaa cagcaagctg
      901 ctgaaagaca aatttgtgtg ccagatccag ctggaaagaa tgaatgaggg ccaggtgcgg
      961 ggagagaaaa accggttcaa gaccgctgat atcatcaagg aaatcatcca gctgctgaat
     1021 gtgcagaaga acttccacca gctggacgag aacttcatca acaagtacat cgaactggtt
     1081 gagatgagac gggaatactt cgagggcccc ggcaagggca gtccatatgg ctgggaaggc
     1141 gaccctaagg cttggtacga gacactgatg ggccactgca cctacttccc agatgagctg
     1201 agaagcgtga aatacgccta cagtgccgac ctgttcaacg ctctgaacga cctgaacaac
     1261 ctggtcatcc aaagagatgg actgtctaag ctcgagtatc atgagaagta tcacatcatc
     1321 gagaacgtgt tcaagcagaa gaagaaacct acactgaagc agatcgccaa tgagatcaat
     1381 gtcaaccctg aagatatcaa gggctacaga atcacaaagt ctggcaagcc ccagtttacc
     1441 gagtttaagc tctaccacga cctgaaaagc gtgctgtttg accagagcat cctggagaac
     1501 gaagacgtgc tggaccagat cgctgagatc ctgaccatct accaggacaa ggatagcatc
     1561 aaatctaagc tgacggaact ggacatcctg ctgaacgagg aagataagga aaacatcgcc
     1621 cagctgactg gctacaccgg gacccaccgg ctcagcctga aatgcatccg gctggtcctg
     1681 gaagagcagt ggtattctag ccggaaccag atggaaatct tcacccacct gaacattaag
     1741 cctaagaaga tcaacctgac agccgccaac aagatcccga aggctatgat cgacgagttc
     1801 atcctgagcc ctgtggtgaa gaggaccttc ggccaggcca ttaaccttat taacaagatc
     1861 atagaaaagt acggcgtgcc tgaagatatc atcatcgagc tggccagaga aaataatagc
     1921 aaggacaagc agaagttcat caatgagatg cagaaaaaga acgagaacac cagaaagaga
     1981 attaacgaaa tcatcggcaa gtatggcaac cagaacgcca agagactggt cgagaagatt
     2041 agactgcacg acgagcagga gggcaagtgc ctgtactcac tggaaagcat ccctctggag
     2101 gacctgctga acaaccccaa ccactacgag gtggaccaca tcattccaag atctgtgtcc
     2161 ttcgacaact cttaccacaa caaagtgctc gtgaagcaga gcgagaactc caaaaaatcc
     2221 aacctgaccc cttaccagta ctttaacagc ggcaagtcca agctctctta caaccagttt
     2281 aaacaacaca tcctgaacct gagcaagtcc caggatagaa tcagcaaaaa aaagaaagag
     2341 tatctgctgg aagaacggga catcaacaag ttcgaggtgc aaaaagagtt catcaataga
     2401 aacctggtgg atacccggta cgccacaaga gagctgacaa actacctgaa ggcctacttc
     2461 agcgccaaca atatgaacgt gaaggtgaaa acgatcaacg gcagcttcac cgattacctg
     2521 cggaaagtgt ggaagtttaa gaaggaacgg aaccacggct acaagcacca cgccgaggac
     2581 gccctgatta tcgctaatgc cgatttcctg ttcaaagaga acaagaagct gaaagccgtg
     2641 aactctgtgc tggaaaaacc tgagatcgag agcaagcagc tggatatcca ggtggatagc
     2701 gaggataact acagcgaaat gttcatcatc cctaagcagg tccaggacat caaggacttc
     2761 agaaacttca agtacagcca cagagtggac aagaagccta acagacggct gatcaacgat
     2821 acactgtaca gcacccggaa gaaggacaac tccacctaca tcgtgcagac catcaaagat
     2881 atctatgcca aagataatac caccctgaag aagcagtttg acaagtcacc cgagaagttc
     2941 ctcatgtacc aacacgatcc gcggaccttc gagaagttgg aagtgatcat gaagcagtac
     3001 gctaatgaga agaatcctct ggccaagtac cacgaggaaa caggcgagta cctgaccaaa
     3061 tacagcaaaa aaaacaacgg ccctatcgtg aaaagcctga agtacattgg aaacaagctg
     3121 ggcagacacc tagatgtgac ccaccagttc aagagcagca ccaagaagtt ggtgaagcgg
     3181 agcatcaagc cttatagatt cgacgtctac ctgaccgaca agggatataa gttcatcacc
     3241 atcagctacc tggacgtgct gaagaaagac aattactact acatacccga acagaagtac
     3301 gacaagctca aactgggcaa ggccatcgac aaaaacgcca agtttatcgc tagcttctac
     3361 aagaatgatc tgatcaagct ggacggcgag atctacaaga tcatcggcgt gagtagcgac
     3421 accagaaaca tgatcgaact ggatctgcct gacatcagat acaaagaata ctgcgagctg
     3481 aacaatatca agggcaaacc tagaatcata aagaccatcg gcaaaaaggt gaatagcatc
     3541 gaaaaactga caaccgacgt gctgggcaac gtgttcacca acacccagta cacaaaacct
     3601 cagctgctgt tcaagcgagg aaattctggc ggctcaaaaa gaaccgccga cggcagcgaa
     3661 ttcgagccca agaagaagag gaaagtcgga agcggagcta ctaacttcag cctgctgaag
     3721 caggctggag acgtggagga gaaccctgga cctatgaccg agtacaagcc cacggtgcgc
     3781 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     3841 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3901 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3961 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     4021 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     4081 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     4141 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     4201 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     4261 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     4321 acctggtgca tgacccgcaa gcccggtgcc tctggtggtt ctcccaagaa gaaaaggaaa
     4381 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     4441 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4501 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4561 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4621 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4681 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4741 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4801 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4861 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4921 gcgcggggag aggcggtttg cgtattgggc gtccgcttcc tcgctcactg actcgctgcg
     4981 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5041 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5101 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5161 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5221 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5281 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5341 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5401 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5461 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5521 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5581 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5641 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5701 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg
     5761 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5821 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5881 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5941 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6001 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6061 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6121 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6181 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6241 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6301 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6361 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6421 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6481 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6541 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6601 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6661 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6721 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6781 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6841 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6901 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     6961 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7021 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     7081 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     7141 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     7201 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     7261 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     7321 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atc
//
