LOCUS       Exported                6688 bp ds-DNA     circular SYN 03-NOV-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6688)
  AUTHORS   Kazuhiko
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Dec 20, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6688
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    30..180
                     /label=Tolmini-L
     promoter        187..205
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    323..457
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        462..479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(544..1467)
                     /codon_start=1
                     /product="zebrafish codon optimized enhanced miRFP2"
                     /label=zemiRFP2
                     /translation="MAEGSVARQPDLLTCEHEEIHLAGSIQPHGTLLVVSEPDHRIIQA
                     SANAAEFLNLGSVLGIPLAEIDGDLLIKILPHLDLTAEGMPVAVRCRIGNPSTEYDGLM
                     HRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGYDRVM
                     VYRFDERGHGEVYSEIHVTGLESYFGNRYPSSLVPQMARRLYVRQRVRVLVDVTYQPVP
                     LEPRLSPLTGRDLDMSGCFLRSMSPTHLQFLKNMGVRATLVVSLVVGGKLWGLVICHHY
                     LPRFIHFELRAICVLLAEAIATRITAL"
     misc_feature    1512..1639
                     /label=CpG free intron
     promoter        complement(1670..1894)
                     
     misc_feature    1895..2320
                     /label=mouse CpG free CMV enh
     misc_feature    2456..2878
                     /label=mouse CpG free CMV enh
     promoter        2879..3103
                     /label=human CpG free EF1 promoter
     misc_feature    3134..3261
                     /label=CpG free synthetic intron
     CDS             3295..4017
                     /codon_start=1
                     /label=mClover3
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK*"
     promoter        complement(4026..4043)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4048..4182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4285..4303)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4310..4510
                     /label=Tol2miniR
     misc_feature    complement(4312..4332)
                     /label=934
     primer_bind     complement(4539..4555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4563..4579
                     /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(4587..4617)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4632..4653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5700..6560)
                     /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(6561..6665)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaatc ccatatgggc agaggtgtaa aaagtactca aaaattttac
       61 tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa aagtatctgg
      121 ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac ttgagtatta
      181 gatctgtaat acgactcact atagggacat agaatgcagg taccgggccc aatgcattgg
      241 cgccgcggcc ggccgcgaat taaaaaacct cccacacctc cccctgaacc tgaaacataa
      301 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
      361 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
      421 gtccaaactc atcaatgtat cttatcatgt ctggatctac gtaatacgac tcactatagt
      481 tctagcctcg agaaggccgg ccataagatc taatggcgcg ccactagttt agatatcgta
      541 acccagagct gtgattcttg tagcgatagc ctcagccagc agcacacaga tagctctcag
      601 ctcgaagtgg atgaatctag gcaggtagtg gtgacagatc accagtcccc acagctttcc
      661 tcccaccacc aggctcacca ccagtgtagc tctcactccc atgttcttca ggaactgcag
      721 gtgtgtaggg ctcatgcttc tcaggaaaca tccgctcatg tccaggtctc ttcctgtcag
      781 agggctcagt ctaggctcca gaggcacagg ctggtatgtc acgtccacca gcactctcac
      841 tctctgtctc acgtacagtc ttctagccat ctgaggcacc aggctgctag ggtatctgtt
      901 tccgaagtag ctctccagtc ctgtcacgtg gatctcgctg tacacctctc cgtgtcctct
      961 ctcgtcgaat ctgtacacca tcactctgtc gtatcctgta cactgctgga acagcagcac
     1021 tgtgtcgtca cacagagctc tcaggcttcc agctgttctg attctctcca gagcaggagc
     1081 cagtgttccg ctcaggtcga tgctaggtcc agctctctcc agctcgatga tcagtcctcc
     1141 ctcaggaggt ctgtgcatca gtccgtcgta ctctgtgcta gggtttccga ttctacatct
     1201 cacagccaca ggcattccct cagctgtcag gtccaggtga ggcaggatct tgatcagcag
     1261 gtctccgtcg atctcagcca gagggattcc cagcacgctt cccaggttca ggaactcagc
     1321 agcgttagcg ctagcctgga tgattctgtg gtcaggctcg ctcaccacca gcagtgttcc
     1381 gtgaggctgg atgcttccag ccaggtggat ctcctcgtgc tcacatgtca ggaggtcagg
     1441 ctgtctagcc acgcttccct cggccatggt ggatatcgtt taaacaattc tggaagctac
     1501 tgtacaccaa cctgtcagga gaggaaagag aagaaggtta gtacaattgt ctagggctgc
     1561 agggttcaca gtgccacttt tcctgcactg ccccacctcc tgcccaccct ttcccaggca
     1621 tagacagtca gtgacttacc aaactcacag gagggagaag gcagaagctt gaatgttcac
     1681 agagactact gcacttatat atggttctcc cccaccctgg ggaaaaaggt ggagccagta
     1741 caccacatca ctttcccagt ttacccaagc cccaccttct ctaggcacca gttcaattgc
     1801 ccacccctcc ccccaacttc tcagggactg tgggccatgt gctctctgcc cactgagggg
     1861 cactcagccc tcaagcatgc tcttctccac tagtcacccc tattgacctt atgtatgtgc
     1921 caataatggg aaaaacccat tgacdsstca ccccctattg accttttgta ctgggcaaaa
     1981 cccaatggaa agtccctatt gactcagtgt acttggctcc aatgggactt tcctgttgat
     2041 tcacccctat tgaccttatg tactgggcaa aacccattgg aaagtcccta atgactcagt
     2101 atacatgtca gtaatgggaa aaacccattg gcttacctcc cattgacctt atgtactggg
     2161 caaaacccat tggaaagtcc ctattgactc aatgtacttg gctccaatgg gactttcctg
     2221 ttgactcacc ccctattgac cttatgtact gggcaaaacc caatggaaag tccctattga
     2281 gtcagtgtac ttggctccaa tgggtttttc ccattgactc ctgcattaat cgatttaaat
     2341 gctagggctg ctctccaatc tctgtcatgg cagggccact ggcatccctg aagttggtgg
     2401 agaccacctc actccactca gcatacagct catccagtcc tcttaattaa tgcaggagtc
     2461 aatgggaaaa acccattgga gccaagtaca ctgactcaat agggactttc cattgggttt
     2521 tgcccagtac ataaggtcaa tagggggtga gtcaacagga aagtcccatt ggagccaagt
     2581 acattgagtc aatagggact ttccaatggg ttttgcccag tacataaggt caatgggagg
     2641 taagccaatg ggtttttccc attactgaca tgtatactga gtcattaggg actttccaat
     2701 gggttttgcc cagtacataa ggtcaatagg ggtgaatcaa caggaaagtc ccattggagc
     2761 caagtacact gagtcaatag ggactttcca ttgggttttg cccagtacaa aaggtcaata
     2821 gggggtgagt caatgggttt ttcccattat tggcacatac ataaggtcaa taggggtgac
     2881 tagtggagaa gagcatgctt gagggctgag tgcccctcag tgggcagaga gcacatggcc
     2941 cacagtccct gagaagttgg ggggaggggt gggcaattga actggtgcct agagaaggtg
     3001 gggcttgggt aaactgggaa agtgatgtgg tgtactggct ccaccttttt ccccagggtg
     3061 ggggagaacc atatataagt gcagtagtct ctgtgaacat tcaagcttct gccttctccc
     3121 tcctgtgagt ttggtaagtc actgactgtc tatgcctggg aaagggtggg caggaggtgg
     3181 ggcagtgcag gaaaagtggc actgtgaacc ctgcagccct agacaattgt actaaccttc
     3241 ttctctttcc tctcctgaca ggttggtgta cagtagcttc cagaattcgc aaacatggtg
     3301 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3361 gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag
     3421 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     3481 accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat gaagcagcac
     3541 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcaag
     3601 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3661 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3721 gagtacaact tcaacagcca ctacgtctat atcacggccg acaagcagaa gaactgcatc
     3781 aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct cgccgaccac
     3841 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3901 agccatcagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3961 gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa gtaataaatc
     4021 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt
     4081 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4141 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4201 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgccacc
     4261 gcggtggagc tccagctttt gttcccttta gtgagggtta attgggccca agtgatctcc
     4321 aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt
     4381 ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag
     4441 taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt
     4501 acacctctgg gtactgtttc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     4561 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4621 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4741 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6661 ccgcgcacat ttccccgaaa agtgccac
//
