LOCUS       Exported                4782 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 4782)
  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..4782
                     /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..1825
                     /label=(1525)_BglII-Clover/Cherry
     misc_feature    1799
                     /label=BglII
     primer_bind     1802..1825
                     /label=(1019)_CCC_FP_F
     CDS             1805..2521
                     /codon_start=1
                     /label=ORF frame 2
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             1805..2521
                     /codon_start=1
                     /label=mClover(A206K)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             complement(1805..2500)
                     /codon_start=1
                     /label=ORF frame 1
                     /translation="MCNPGGGHELQQDHVIALLVGVFAQLGLMAQVVVVGQQHGAVADG
                     GVLLVVVGELHAAVLNVVADLEVSLDAVLLLVGRDIDVVAVEVVLQLVPQDVAVLLEVD
                     ALQLDAVHQGVALELHLGAGLVGTVVLERDGALLDVAFGHGGLEEVVLLHVVGVAAEAG
                     HAVAEGGHEGGPGHGQLAGGADELQGQLAVGGIALALAADAELVAVYVAVQLDQDGHHP
                     GEQLLALAHH"
     primer_bind     1805..1825
                     /label=(996)_Linker_Clover_5'_BamHI_SacI
     gene            1808..2521
                     /gene="EGFP (variant)"
                     /label=EGFP (variant)
     gene            1808..2521
                     /gene="ECFP (variant)"
                     /label=ECFP (variant)
     misc_feature    complement(1850..1871)
                     /label=EGFP_N_primer
     primer_bind     complement(2364..2383)
                     /label=(534) GFP seq 3
     primer_bind     complement(2408..2440)
                     /label=(1018)_mClover(A206K)_SDM_R
     primer_bind     2408..2440
                     /label=(1017)_mClover(A206K)_SDM_F
     misc_feature    2423..2425
                     /label=A206K
     misc_feature    2458..2479
                     /label=EGFP_C_primer
     primer_bind     complement(2501..2545)
                     /label=(1212) Clover/Cherry-linker-BamHI
     primer_bind     complement(2502..2521)
                     /label=(1020)_CCC_mClo_TAA_NotI_R
     CDS             2522..2539
                     /codon_start=1
                     /label=Linker
                     /translation="GAGAGA"
     primer_bind     2528..2537
                     /label=(965)_pAID_Linker_5'_BamHI_SacI
     promoter        complement(2594..2612)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2629..2647)
                     /label=(2) M13 reverse primer
     primer_bind     complement(2633..2649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2657..2673
                     /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(2681..2711)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2726..2747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3035..3623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3794..4654)
                     /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(4655..4759)
                     /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 atctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1861 ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac
     1921 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1981 caccctcgtg accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat
     2041 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2101 ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac
     2161 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2221 gcacaagctg gagtacaact tcaacagcca caacgtctat atcacggccg acaagcagaa
     2281 gaacggcatc aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct
     2341 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2401 ccactacctg agccatcagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     2461 ggtcctgctg gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa
     2521 gggcgctggt gcaggcgccg gatccactag ttctagagcg gccgccaccg cggtggagct
     2581 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     2641 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2701 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2761 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2821 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2881 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2941 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3001 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3061 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3121 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3181 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3241 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3301 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3361 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3421 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3481 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3541 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3601 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3661 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3721 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3781 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3841 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3901 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3961 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4021 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4081 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4141 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4201 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4261 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4321 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4381 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4441 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4501 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4561 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4621 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4681 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4741 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
