LOCUS       Exported                4188 bp ds-DNA     circular SYN 11-DEC-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4188)
  AUTHORS   Toyoaki Natsume
  TITLE     Direct Submission
  JOURNAL   Exported Dec 11, 2018 from SnapGene Viewer 4.2.9
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4188
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    701..706
                     /label=kozak seq
     CDS             707..1729
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1742..1798
                     /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"
     primer_bind     1799..1821
                     /label=(1526)_BglII-S-Tag-3xFLAG
     misc_feature    1799
                     /label=BglII
     CDS             1805..1849
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1862..1927
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1906..1952)
                     /label=(1527)_S-Tag-3xFLAG-linker-BamHI
     CDS             1928..1945
                     /codon_start=1
                     /label=Linker
                     /translation="GAGAGA"
     promoter        complement(2000..2018)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2035..2053)
                     /label=(2) M13 reverse primer
     primer_bind     complement(2039..2055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2063..2079
                     /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(2087..2117)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2132..2153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2441..3029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3200..4060)
                     /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(4061..4165)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gccaccatga aaaagcctga
      721 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
      781 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
      841 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
      901 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
      961 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     1021 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     1081 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     1141 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     1201 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     1261 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     1321 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     1381 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     1441 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     1501 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     1561 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     1621 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     1681 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat caggaagcgg
     1741 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctag
     1801 atctaaggaa accgccgctg caaaattcga acgccagcac atggacagcc gatcccgggc
     1861 tgactacaaa gaccatgacg gtgattataa agatcatgac atcgactaca aggatgacga
     1921 tgacaagggc gctggtgcag gcgccggatc cactagttct agagcggccg ccaccgcggt
     1981 ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     2041 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2101 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2161 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2221 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2281 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2341 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2401 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2461 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2521 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2581 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2641 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2701 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2761 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2821 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2881 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2941 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3001 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3061 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3121 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3181 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3241 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3301 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3361 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3421 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3481 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3541 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3601 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3661 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3721 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3781 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3841 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3901 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3961 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4021 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4081 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4141 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
