LOCUS       sequence-449656-        4499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Zeocin resistance marker followed by miR-430 target 
            site insertion with mismatches at position 10 and 11 in zebrafish 
            embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4499)
  AUTHORS   Chen GR, Sive H, Bartel DP
  TITLE     A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially
            Rescues Severely Impaired Cleavage Found in Fish.
  JOURNAL   Mol Cell. 2017 Dec 21;68(6):1095-1107.e5. doi: 
            10.1016/j.molcel.2017.11.032.
  PUBMED    29272705
REFERENCE   2  (bases 1 to 4499)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2017.11"; journalName: "Mol Cell"; date: 
            "2017-12-21- 21"; volume: "68"; issue: "6"; pages: "1095-1107.e5"
COMMENT     Sequence Label: pCS2+-Zeo-miR-430-10-11 mm
FEATURES             Location/Qualifiers
     source          1..4499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             106..480
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    551..685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(575..594)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     629..648
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(798..818)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(798..816)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(837..853)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(837..853)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(850..872)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    861..877
                     /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(join(885..891,892..909,910..915))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    930..951
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1068..1085)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1239..1827)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1319..1338)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1998..2789,2790..2858))
                     /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"
     primer_bind     2621..2640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2859..2963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2989..3444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3338..3357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3515..4499
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca ggatcatggc caagttgacc
      121 agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac
      181 cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac
      241 gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg
      301 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac
      361 ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag
      421 ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga
      481 ctcgagcctc tagaaataag ctaccccaac ttcttagcac tttataagct atagtgagtc
      541 gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
      601 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
      661 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
      721 tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc attgggcccg
      781 gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
      841 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccggg
      901 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
      961 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1021 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1081 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1141 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1201 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1261 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1321 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1381 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1441 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1501 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1561 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1621 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1681 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1741 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1801 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1861 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1921 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1981 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2041 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2101 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2161 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2221 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2281 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2341 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2401 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2461 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2521 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2581 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2641 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2701 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2761 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2821 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2881 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2941 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     3001 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3061 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3421 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     3481 ggcgatcggt gcgggcctct tcgctattac gccagtcgac cgccaattca atatggcgta
     3541 tatggactca tgccaattca atatggtgga tctggacctg tgccaattca atatggcgta
     3601 tatggactcg tgccaattca atatggtgga tctggacccc agccaattca atatggcgga
     3661 cttggcacca tgccaattca atatggcgga cctggcactg tgccaactgg ggaggggtct
     3721 acttggcacg gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg
     3781 aattggcatg gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg
     3841 caatatccaa tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg
     3901 gccctatgcc atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc
     3961 aatgggcggt cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc
     4021 attgacgtca atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat
     4081 tgacgtatat ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa
     4141 tgcccattga cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca
     4201 ttcatatccg ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag
     4261 tacatcaata tctattaata gtaacttggc aagtacatta ctattggaag tacgccaggg
     4321 tacattggca gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag
     4381 tacatcccca ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt
     4441 aaatgggcgg taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaac
//
