LOCUS       Exported                7578 bp ds-DNA     circular SYN 10-SEP-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7578)
  AUTHORS   Enzo Mameli
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Sep 10, 2021 from SnapGene 5.1.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7578
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             201..1061
                     /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"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2276..2309
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        2319..2518
                     /label=U6 (AAEL028972)
                     /note="promoter from AAEL028972"
     misc_feature    2414
                     /label=C>G
                     /note="C>G to disrupt kpnI site, position not conserved "
     misc_RNA        2537..2612
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2621..2671
                     /label=3' end flanking AAEL028972
                     /note="contig NIGP01001856.1 50092..50998(1)"
     misc_feature    2623
                     /label=A>G
                     /note="A>G to disrupt bstbI restriction site, G is 
                     morefrequent in the same position in U6 more expressed"
     misc_feature    2672..2794
                     /label=3'end snRNAU696AB Dmel
     promoter        2853..5362
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             5396..6112
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             6116..6169
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6194..6793
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6820..6941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    7135..7168
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(7184..7200)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc cccgggtgcc
     2281 agggcgtgcc cttgggctcc ccgggcgcgt acttcgaacg tcggctggct ggattccaat
     2341 ttgaatattg gctaattgga agagatggaa gtttttgaat ggatgattga ataattgaag
     2401 cgactccggg tacgtgtttg taagctctgc aacagtgaca tagatttgtg tcagtccatc
     2461 actagaatca aatcaacttg tacttgctat ataaatggct aggactagcg gaagatttgg
     2521 gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2581 atcaacttga aaaagtggca ccgagtcggt gctttttttt cggatttgtc atgcattgaa
     2641 ctaagttagt tttcacagta ataaaacctc attttttgcc tacctggagc ctgagagttg
     2701 ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca gtatttatta tttttcatca
     2761 atatgtattc aatttggtat gtatttagta attgggtacc cagcatgcaa ttctatattc
     2821 taaaaacaca aatgatactt ctaaaaaaaa atcatgaatg gcatcaactc tgaatcaaat
     2881 ctttgcagat gcacctactt ctcatttcca ctgtcacatc atttttccag atctcgctgc
     2941 ctgttatgtg gcccacaaac caagacacgt tttatggcca ttaaagctgg ctgatcgtcg
     3001 ccaaacacca aatacataat gaatatgtac acattcgaga aagaagcgat caaagaagca
     3061 tcttcgggcg gagtaggaga atgcggagga gaaggagaac gagctgatct agtatctctc
     3121 cacaatccaa tgccaactga ccaactggcc atattcggag caatttgaag ccaatttcca
     3181 tcgcctggcg atcgctccat tcttggctat atgtttttca ccgttacccg gggccatttt
     3241 caaagactcg tcggcaagat aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga
     3301 atgctgagga atttcgcgat gacgtcggcg agtattttga agaatgagaa taatttgtat
     3361 ttatacgaaa atcagttagt ggaattttct acaaaaacat gttatctata gataattttg
     3421 ttgcaaaata tgttgactat gacaaagatt gtatgtatat acctttaatg tattctcatt
     3481 ttcttatgta tttataatgg caatgatgat actgatgata ttttaagatg atgccagacc
     3541 aaaaggcttg aatttctgcg tcttttgccg aacgcagtgc atgtgcaatt gttgtttttt
     3601 ggaatattca attttcggac tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc
     3661 aaagtaaagc caaaaaagcg agatggcaat accaaatgcg gcaaaacggt agtggaagga
     3721 aaggggtgcg gggcagcgga aggaagggtg gggcggggcg tggcggggtc tgtggctggg
     3781 cgcgacgtca ccgacgttgg agccactcct ttgaccatgt gtgcgtgtgt gtattattcg
     3841 tgtctcgcca ctcgccggtt gtttttttct ttttatgctg cgctctctct agcgccatct
     3901 cgcttacgca tgctcaacgc accgcatgtt gccgtttcct tttatgcgtc attttggctc
     3961 gaaataggca attatttaaa caaagattag tcaacgaaaa cgctaaaata aataagtcta
     4021 caatatggtt acttattgcc atgtgtgtgc agccaacgat agcaacaaaa gcaacaacac
     4081 aggtggcttt ccctctttca ctttttgttt gcaagccgcg tgcgagcaag acggcacgac
     4141 cggcaaacgc aattacgctg acaaagagca gacgaagttt tggcgaaaaa catcaaggcg
     4201 cctgatacga atgcatttgc aataacaatt gcgatattta atattgttta tgaagctgtt
     4261 tgacttcaaa acacacaaaa aaaaaaataa aacaaattat ttgaaagaga attaggaatc
     4321 ggacgcttat cgttagggta acaacaagaa atgcttactg agtcacagcc tctggaaaac
     4381 tgccgcaagc cagagagaga gagaaaaaga gggagagcag cttagaccgc atgtgcttgt
     4441 gtgtgaggcg tctctctctt cgtctctgtt gcgcaaacgc atagactgca ctgagaaaat
     4501 cgattaccta ttttttatga atgaatattt gcactattac tattcaaaac tattaagata
     4561 gcaatcacat tcaatagcca aatactatac cacctgagcg atgcaacgaa atgatcaatt
     4621 tgagcaaaaa tgctgcatat ttaggacggc atcattatag aaatgcttct tgctgtgtac
     4681 ttttctctcg tctggcagct gtttcgccgt tattgttaaa accggcttaa gttaggtgtg
     4741 ttttctacga ctagtgaatg ccctactaga agatgtgtgt tgcacaaaat gtccctggaa
     4801 taaccaattt gaagtgcaga tagcagtaaa cgtaagctaa tatgaatatt atttaactgt
     4861 aatgttttaa tatcgctgga cattactaat aaacccacta taaacacatg tacatatgta
     4921 tgttttggca tacaatgagt agttggggaa aaaatgtgta aaagcaccgt gaccatcaca
     4981 gcataaagat aaccagctga agtatcgaat atgagtaacc cccaaattga atcacatgcc
     5041 gcaactgata ggacccatgg aagtacactc ttcatggcga tatacaagac acacacaagc
     5101 acgaacaccc agttgcggag gaaattctcc gtaaatgaaa acccaatcgg cgaacaattc
     5161 atacccatat atggtaaaag ttttgaacgc gacttgagag cggagagcat tgcggctgat
     5221 aaggttttag cgctaagcgg gctttataaa acgggctgcg ggaccagttt tcatatcact
     5281 accgtttgag ttcttgtgct gtgtggatac tcctcccgac acaaagccgc tccatcagcc
     5341 agcagtcgtc taatccagag accccggatc cagatgaatt cgcccttgtt taaacatggt
     5401 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     5461 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa
     5521 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     5581 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     5641 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     5701 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa
     5761 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     5821 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat
     5881 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     5941 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct
     6001 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     6061 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agcttgaggg
     6121 cagaggaagc cttctaacat gcggtgacgt ggaggagaat cccggccctg ctagcggtag
     6181 cggcagcggt agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     6241 cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     6301 cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     6361 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     6421 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     6481 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     6541 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     6601 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     6661 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     6721 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     6781 gcccggtgcc tgactcgagg gatccgaatt cagacatgat aagatacatt gatgagtttg
     6841 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     6901 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     6961 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     7021 acaaatgtgg tatggctgat tatgatcagt cgaccgatgc ccttgagagc cttcaaccca
     7081 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat ggtacgcgcc
     7141 cggggagccc aagggcacgc cctggcaccc gaagggcgaa ttcactggcc gtcgttttac
     7201 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     7261 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     7321 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     7381 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7441 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7561 catcaccgaa acgcgcga
//
