LOCUS       Exported                6685 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 6685)
  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..6685
                     /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(523..1464)
                     /codon_start=1
                     /product="zebrafish codon optimized miRFP"
                     /label=zmiRFP
                     /translation="MVAGHASGSPDFGTADPSDCEREEIHLAGSIQPHGTLLVVSEPDH
                     RIIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YDGLMHRPPEGGLIIELERAGPPIDLSGTLAPALERIRTAGSLRALCDDTALLFQQCTG
                     YDRVMVYRFDEQGHGEVYSEIHVTGLESYFGNRYPSSLVPQMARRLYERQRVRVLVDVS
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPTHLQFLKNMGVRATLVVSLVVGGKLWGLV
                     ICHHYLPRFIHFELRAICELLAEAIATRITAL"
     misc_feature    1509..1636
                     /label=CpG free intron
     promoter        complement(1667..1891)
                     
     misc_feature    1892..2317
                     /label=mouse CpG free CMV enh
     misc_feature    2453..2875
                     /label=mouse CpG free CMV enh
     promoter        2876..3100
                     /label=human CpG free EF1 promoter
     misc_feature    3131..3258
                     /label=CpG free synthetic intron
     CDS             3292..4014
                     /codon_start=1
                     /label=mClover3
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK*"
     promoter        complement(4023..4040)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4045..4179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4282..4300)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4307..4507
                     /label=Tol2miniR
     misc_feature    complement(4309..4329)
                     /label=934
     primer_bind     complement(4536..4552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4560..4576
                     /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(4584..4614)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4629..4650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4938..5526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5697..6557)
                     /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(6558..6662)
                     /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 cctcacagag ctgtgattct
      541 tgtagcgata gcctcagcca gcagctcaca gatagctctc agctcgaagt ggatgaatct
      601 aggcaggtag tggtgacaga tcaccagtcc ccacagcttt cctcccacca ccaggctcac
      661 caccagtgta gctctcactc ccatgttctt caggaactgc aggtgtgtag ggctcatgct
      721 tctcaggaaa catccgctca tgtccaggtc tcttcctgtc agagggctca gtctaggctc
      781 cagaggcaca ggctggtagc tcacgtccac cagcactctc actctctgtc tctcgtacag
      841 tcttctagcc atctgaggca ccaggctgct agggtatctg tttccgaagt agctctccag
      901 tcctgtcacg tggatctcgc tgtacacctc tccgtgtccc tgctcgtcga atctgtacac
      961 catcactctg tcgtatcctg tacactgctg gaacagcaga gctgtgtcgt cacacagagc
     1021 tctcaggctt ccagctgttc tgattctctc cagagcagga gccagtgttc cgctcaggtc
     1081 gataggaggt ccagctctct ccagctcgat gatcagtcct ccctcaggag gtctgtgcat
     1141 cagtccgtcg tactctgtgc tagggtttcc gattctacat ctcacagcca caggcattcc
     1201 ctcagctgta gggtccaggt gaggcaggat cttgatcagc aggtctccgt cgatctcagc
     1261 cagaggcact cccagcacgc ttcccaggtt caggaactca gcagcgttag cgctagcctg
     1321 gatgattctg tggtcaggct cgctcaccac cagcagtgtt ccgtgaggct ggatgcttcc
     1381 agccaggtgg atctcctctc tctcacagtc gctagggtca gctgttccga agtcagggct
     1441 tccgctagcg tgtccagcca ccatggtgga tatcgtttaa acaattctgg aagctactgt
     1501 acaccaacct gtcaggagag gaaagagaag aaggttagta caattgtcta gggctgcagg
     1561 gttcacagtg ccacttttcc tgcactgccc cacctcctgc ccaccctttc ccaggcatag
     1621 acagtcagtg acttaccaaa ctcacaggag ggagaaggca gaagcttgaa tgttcacaga
     1681 gactactgca cttatatatg gttctccccc accctgggga aaaaggtgga gccagtacac
     1741 cacatcactt tcccagttta cccaagcccc accttctcta ggcaccagtt caattgccca
     1801 cccctccccc caacttctca gggactgtgg gccatgtgct ctctgcccac tgaggggcac
     1861 tcagccctca agcatgctct tctccactag tcacccctat tgaccttatg tatgtgccaa
     1921 taatgggaaa aacccattga cdsstcaccc cctattgacc ttttgtactg ggcaaaaccc
     1981 aatggaaagt ccctattgac tcagtgtact tggctccaat gggactttcc tgttgattca
     2041 cccctattga ccttatgtac tgggcaaaac ccattggaaa gtccctaatg actcagtata
     2101 catgtcagta atgggaaaaa cccattggct tacctcccat tgaccttatg tactgggcaa
     2161 aacccattgg aaagtcccta ttgactcaat gtacttggct ccaatgggac tttcctgttg
     2221 actcaccccc tattgacctt atgtactggg caaaacccaa tggaaagtcc ctattgagtc
     2281 agtgtacttg gctccaatgg gtttttccca ttgactcctg cattaatcga tttaaatgct
     2341 agggctgctc tccaatctct gtcatggcag ggccactggc atccctgaag ttggtggaga
     2401 ccacctcact ccactcagca tacagctcat ccagtcctct taattaatgc aggagtcaat
     2461 gggaaaaacc cattggagcc aagtacactg actcaatagg gactttccat tgggttttgc
     2521 ccagtacata aggtcaatag ggggtgagtc aacaggaaag tcccattgga gccaagtaca
     2581 ttgagtcaat agggactttc caatgggttt tgcccagtac ataaggtcaa tgggaggtaa
     2641 gccaatgggt ttttcccatt actgacatgt atactgagtc attagggact ttccaatggg
     2701 ttttgcccag tacataaggt caataggggt gaatcaacag gaaagtccca ttggagccaa
     2761 gtacactgag tcaataggga ctttccattg ggttttgccc agtacaaaag gtcaataggg
     2821 ggtgagtcaa tgggtttttc ccattattgg cacatacata aggtcaatag gggtgactag
     2881 tggagaagag catgcttgag ggctgagtgc ccctcagtgg gcagagagca catggcccac
     2941 agtccctgag aagttggggg gaggggtggg caattgaact ggtgcctaga gaaggtgggg
     3001 cttgggtaaa ctgggaaagt gatgtggtgt actggctcca cctttttccc cagggtgggg
     3061 gagaaccata tataagtgca gtagtctctg tgaacattca agcttctgcc ttctccctcc
     3121 tgtgagtttg gtaagtcact gactgtctat gcctgggaaa gggtgggcag gaggtggggc
     3181 agtgcaggaa aagtggcact gtgaaccctg cagccctaga caattgtact aaccttcttc
     3241 tctttcctct cctgacaggt tggtgtacag tagcttccag aattcgcaaa catggtgagc
     3301 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3361 aacggccaca agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg
     3421 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3481 accttcggct acggcgtggc ctgcttcagc cgctaccccg accacatgaa gcagcacgac
     3541 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc tttcaaggac
     3601 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3661 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3721 tacaacttca acagccacta cgtctatatc acggccgaca agcagaagaa ctgcatcaag
     3781 gctaacttca agatccgcca caacgttgag gacggcagcg tgcagctcgc cgaccactac
     3841 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3901 catcagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3961 ttcgtgaccg ccgccgggat tacacatggc atggacgagc tgtacaagta ataaatctag
     4021 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     4081 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4141 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4201 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgccaccgcg
     4261 gtggagctcc agcttttgtt ccctttagtg agggttaatt gggcccaagt gatctccaaa
     4321 aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta
     4381 aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa
     4441 aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca
     4501 cctctgggta ctgtttcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4561 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4621 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4681 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4741 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4801 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4861 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4921 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4981 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5041 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5101 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5161 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5221 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5281 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5341 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5401 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5461 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5521 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5581 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5641 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5701 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5761 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5821 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5881 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5941 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6001 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6061 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6121 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6181 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6241 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6301 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6361 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6421 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6481 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6541 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6601 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6661 cgcacatttc cccgaaaagt gccac
//
