LOCUS       sequence-449654-        4262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Zeocin resistance marker in zebrafish embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4262)
  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 4262)
  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
FEATURES             Location/Qualifiers
     source          1..4262
                     /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    518..652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(542..561)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     596..615
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(765..785)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(765..783)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(804..820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(804..820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(817..839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    828..844
                     /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(852..858,859..876,877..882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    897..918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1035..1052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1206..1794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1286..1305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1965..2756,2757..2825))
                     /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     2588..2607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2826..2930)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2956..3411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3093..3114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3305..3324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
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 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat
      541 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
      601 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
      661 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc
      721 ggccgcggcg ccaatgcatt gggcccggta cccagctttt gttcccttta gtgagggtta
      781 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      841 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
      901 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
      961 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1021 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1081 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1141 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1201 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1261 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1321 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1381 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1441 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1501 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1561 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1621 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1681 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1741 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1801 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1861 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1921 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1981 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2041 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2101 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2161 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2221 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2281 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2341 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2401 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2461 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2521 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2581 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2641 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2701 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2761 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2821 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2881 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2941 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     3001 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3061 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     3121 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3181 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3241 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3301 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3361 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     3421 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3481 agtcgaccgc caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct
     3541 ggacctgtgc caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct
     3601 ggaccccagc caattcaata tggcggactt ggcaccatgc caattcaata tggcggacct
     3661 ggcactgtgc caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct
     3721 tggccctgtg ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt
     3781 ggctatatgc caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct
     3841 attggccagg ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt
     3901 ttcctattga cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt
     3961 cctaataccg cccatagcca ctcccccatt gacgtcaatg acggtaaatg gcccacttgg
     4021 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca
     4081 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
     4141 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
     4201 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
     4261 ac
//
