LOCUS       Exported                6703 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 6703)
  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..6703
                     /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..1482)
                     /codon_start=1
                     /product="zebrafish codon optimized miRFP2"
                     /label=zmiRFP2
                     /translation="MVAGHASGSPDFGTASPSDCEHEEIHLAGSIQPHGTLLVVSEPDH
                     RIIQASANAAEFLNLGSVLGIPLAEIDGDLLIKILPHLDLTAEGMPVAVRCRIGNPSTE
                     YDGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDERGHGEVYSEIHVTGLESYFGNRYPSSLVPQMARRLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPTHLQFLKNMGVRATLVVSLVVGGKLWGLV
                     ICHHYLPRFIHFELRAICVLLAEAIATRITAL"
     misc_feature    1527..1654
                     /label=CpG free intron
     promoter        complement(1685..1909)
                     
     misc_feature    1910..2335
                     /label=mouse CpG free CMV enh
     misc_feature    2471..2893
                     /label=mouse CpG free CMV enh
     promoter        2894..3118
                     /label=human CpG free EF1 promoter
     misc_feature    3149..3276
                     /label=CpG free synthetic intron
     CDS             3310..4032
                     /codon_start=1
                     /label=mClover3
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK*"
     promoter        complement(4041..4058)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4063..4197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4300..4318)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4325..4525
                     /label=Tol2miniR
     misc_feature    complement(4327..4347)
                     /label=934
     primer_bind     complement(4554..4570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4578..4594
                     /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(4602..4632)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4647..4668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4956..5544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5715..6575)
                     /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(6576..6680)
                     /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 tcacagtcgc tagggctagc
     1441 tgttccgaag tcagggcttc cgctagcgtg tccagccacc atggtggata tcgtttaaac
     1501 aattctggaa gctactgtac accaacctgt caggagagga aagagaagaa ggttagtaca
     1561 attgtctagg gctgcagggt tcacagtgcc acttttcctg cactgcccca cctcctgccc
     1621 accctttccc aggcatagac agtcagtgac ttaccaaact cacaggaggg agaaggcaga
     1681 agcttgaatg ttcacagaga ctactgcact tatatatggt tctcccccac cctggggaaa
     1741 aaggtggagc cagtacacca catcactttc ccagtttacc caagccccac cttctctagg
     1801 caccagttca attgcccacc cctcccccca acttctcagg gactgtgggc catgtgctct
     1861 ctgcccactg aggggcactc agccctcaag catgctcttc tccactagtc acccctattg
     1921 accttatgta tgtgccaata atgggaaaaa cccattgacd sstcaccccc tattgacctt
     1981 ttgtactggg caaaacccaa tggaaagtcc ctattgactc agtgtacttg gctccaatgg
     2041 gactttcctg ttgattcacc cctattgacc ttatgtactg ggcaaaaccc attggaaagt
     2101 ccctaatgac tcagtataca tgtcagtaat gggaaaaacc cattggctta cctcccattg
     2161 accttatgta ctgggcaaaa cccattggaa agtccctatt gactcaatgt acttggctcc
     2221 aatgggactt tcctgttgac tcacccccta ttgaccttat gtactgggca aaacccaatg
     2281 gaaagtccct attgagtcag tgtacttggc tccaatgggt ttttcccatt gactcctgca
     2341 ttaatcgatt taaatgctag ggctgctctc caatctctgt catggcaggg ccactggcat
     2401 ccctgaagtt ggtggagacc acctcactcc actcagcata cagctcatcc agtcctctta
     2461 attaatgcag gagtcaatgg gaaaaaccca ttggagccaa gtacactgac tcaataggga
     2521 ctttccattg ggttttgccc agtacataag gtcaataggg ggtgagtcaa caggaaagtc
     2581 ccattggagc caagtacatt gagtcaatag ggactttcca atgggttttg cccagtacat
     2641 aaggtcaatg ggaggtaagc caatgggttt ttcccattac tgacatgtat actgagtcat
     2701 tagggacttt ccaatgggtt ttgcccagta cataaggtca ataggggtga atcaacagga
     2761 aagtcccatt ggagccaagt acactgagtc aatagggact ttccattggg ttttgcccag
     2821 tacaaaaggt caataggggg tgagtcaatg ggtttttccc attattggca catacataag
     2881 gtcaataggg gtgactagtg gagaagagca tgcttgaggg ctgagtgccc ctcagtgggc
     2941 agagagcaca tggcccacag tccctgagaa gttgggggga ggggtgggca attgaactgg
     3001 tgcctagaga aggtggggct tgggtaaact gggaaagtga tgtggtgtac tggctccacc
     3061 tttttcccca gggtggggga gaaccatata taagtgcagt agtctctgtg aacattcaag
     3121 cttctgcctt ctccctcctg tgagtttggt aagtcactga ctgtctatgc ctgggaaagg
     3181 gtgggcagga ggtggggcag tgcaggaaaa gtggcactgt gaaccctgca gccctagaca
     3241 attgtactaa ccttcttctc tttcctctcc tgacaggttg gtgtacagta gcttccagaa
     3301 ttcgcaaaca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     3361 gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg cgagggcgat
     3421 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     3481 tggcccaccc tcgtgaccac cttcggctac ggcgtggcct gcttcagccg ctaccccgac
     3541 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     3601 accatctctt tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc
     3661 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     3721 ctggggcaca agctggagta caacttcaac agccactacg tctatatcac ggccgacaag
     3781 cagaagaact gcatcaaggc taacttcaag atccgccaca acgttgagga cggcagcgtg
     3841 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     3901 gacaaccact acctgagcca tcagtccaag ctgagcaaag accccaacga gaagcgcgat
     3961 cacatggtcc tgctggagtt cgtgaccgcc gccgggatta cacatggcat ggacgagctg
     4021 tacaagtaat aaatctagaa ctatagtgag tcgtattacg tagatccaga catgataaga
     4081 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     4141 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     4201 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     4261 ttcgcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgg
     4321 gcccaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg taaggagtaa
     4381 aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt gatttttaat
     4441 tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa tcaagtaaaa
     4501 ttactcaagt actttacacc tctgggtact gtttcgcgct tggcgtaatc atggtcatag
     4561 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4621 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4681 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4741 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4801 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4861 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4921 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4981 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5041 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5101 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5161 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5221 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5281 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5341 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5401 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5461 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5521 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5581 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5641 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5701 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5761 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5821 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5881 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5941 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6001 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6061 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6121 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6181 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6241 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6301 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6361 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6421 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6481 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6541 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6601 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6661 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
