LOCUS       sequence-7294-ex        8595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8595)
  AUTHORS   Sigova A, Rhind N, Zamore PD
  TITLE     A single Argonaute protein mediates both transcriptional and 
            posttranscriptional silencing in Schizosaccharomyces pombe.
  JOURNAL   Genes Dev. 2004 Oct 1. 18(19):2359-67.
  PUBMED    15371329
REFERENCE   2  (bases 1 to 8595)
  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"; journalName: "Genes Dev. 
            2004 Oct 1. 18(19):2359-67."
COMMENT     Sequence Label: pAS1
FEATURES             Location/Qualifiers
     source          1..8595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             8..718
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLDTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             complement(795..1505)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLDTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2522..2539)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2522..2538)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2531..2553)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2751..3206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2838..2857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3048..3069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3223..3242)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3342..3364
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3402..3420)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3488..3592
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3593..3661,3662..4453)
                     /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     complement(3811..3830)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4624..5212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5113..5132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5366..5383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5500..5521
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5536..5541,5542..5559,5560..5566)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5574..5590
                     /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)."
     primer_bind     5579..5601
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5598..5614
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5598..5614
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(6063..6857)
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     promoter        join(7433..8595,1..2)
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
ORIGIN
        1 catatggagt aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
       61 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata
      121 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac
      181 acttgacact actctcactt atggtgttca atgcttttca agatacccag atcacatgaa
      241 acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt
      301 tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct
      361 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
      421 caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa
      481 tggaatcaaa gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc
      541 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
      601 ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt
      661 ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata
      721 aaatcaggtg tgttggaact tttttcaaac cttactaaac attgaaacta attggtaaag
      781 atagaggcta gctatttgta tagttcatcc atgccatgtg taatcccagc agctgttaca
      841 aactcaagaa ggaccatgtg gtctctcttt tcgttgggat ctttcgaaag ggcagattgt
      901 gtggacaggt aatggttgtc tggtaaaagg acagggccat cgccaattgg agtattttgt
      961 tgataatggt ctgctagttg aacgcttcca tcttcaatgt tgtgtctaat tttgaagtta
     1021 actttgattc cattcttttg tttgtctgcc atgatgtata cattgtgtga gttatagttg
     1081 tattccaatt tgtgtccaag aatgtttcca tcttctttaa aatcaatacc ttttaactcg
     1141 attctattaa caagggtatc accttcaaac ttgacttcag cacgtgtctt gtagttcccg
     1201 tcatctttga aaaatatagt tctttcctgt acataacctt cgggcatggc actcttgaaa
     1261 aagtcatgct gtttcatgtg atctgggtat cttgaaaagc attgaacacc ataagtgaga
     1321 gtagtgtcaa gtgttggcca tggaacaggt agttttccag tagtgcaaat aaatttaagg
     1381 gtaagttttc cgtatgttgc atcaccttca ccctctccac tgacagaaaa tttgtgccca
     1441 ttaacatcac catctaattc aacaagaatt gggacaactc cagtgaaaag ttcttctcct
     1501 ttactcggat ccccgggtaa aaggaatgtc tcccttgcca gtactgctag ggtttttctt
     1561 tcaaactatg gaagcccatt caagctgcat attacgattt tgtttttcgc ttttagaaag
     1621 tggtttagat gagataatag aaaaattctt gatctccgac aacgagtact tttatttttt
     1681 ttgctaatca ctttactcaa tattagctcg aaatcgtaga aacgtagacg ggtgcgggat
     1741 accgagtggt gtagttaaga atttttataa accacgtggc ccaaaaatat gaacccaaaa
     1801 cgtttataca tgagtatact ttaagaaggc tatacccctt cgtgttagat gtagttttag
     1861 ctacccaacc cgagtctatg agcttgactt cagatgtaga aggcattaaa tcgttttgaa
     1921 tattaattaa aaaacgatga aaattaaata tttaaaagca atcatacgct gaaaatttag
     1981 tgctgtggct aatccttcaa catggaaatg ccataaaagt gactttgaca aaaaaaaaag
     2041 tatatacagg tagtaaactc atctacttca ttgactttgt ttacagcatg tggaaggagg
     2101 aatatttatt gctaaatcgt agtttaacat tcaataagta atactattga aattcgacaa
     2161 gattggccgc atggatgaaa aagaggcatt ttgctttggg agaattagtt caaattagaa
     2221 ctgaaaaaaa aaactttacg aggcaaaaat gtcggattga gatcgtaaaa gttcgctcgt
     2281 cgtcttttgc tttgtgattg ttttcatgga tacatcttgc tggatattta aattttagta
     2341 ctatgtataa gatattctat aaatgtttta tcacccaaac ctgttagcgc cttcttaatt
     2401 ctattcaatc tggcttttgc tctgagacta cttcttggac tttcactact tgttagttat
     2461 acggaatttg tgtaattaga agtgaaataa tcctttctat tagtaatgcg agctcgaatt
     2521 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2581 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2641 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     2701 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     2761 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2821 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2881 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2941 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     3001 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3061 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     3121 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3181 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     3241 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3301 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3361 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     3421 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3541 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3601 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3661 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3721 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3781 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3841 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3901 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3961 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4021 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4081 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4141 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4201 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4261 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4321 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4381 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4441 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4501 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4561 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4621 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4681 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4741 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4801 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4861 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4921 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4981 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5041 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5101 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5161 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5221 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5281 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5341 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5401 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5461 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     5521 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5581 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgtgatatt
     5641 gacgaaactt tttgacatct aatttattct gttccaacac caatgtttat aaccaagttt
     5701 tatcttgttt gtctacatgg tattttacat tcatctacat acatctttca ttggctttgt
     5761 acatagttat cattacaagt ctaaaaaaat tcactctttt cttattcaat gtcaatccaa
     5821 gagaaaagat tgtggtaatg ttgtaggagc atgtttaata aattactata gcaaattact
     5881 ttttattccc aaggtgttta tctataatag ttaatatttt agtcgctaca taaaatttta
     5941 ccaaagagta cttgtatact aattctaaat gccttctgac ataaaacgcc taggaaaaca
     6001 aacgcaaaca aggcatcgac tttttcaata accaaccaaa aaaattttac attagtcttt
     6061 ttttaatgct gagaaagtct ttgctgatat gccttccaac cagcttctct atatctcttg
     6121 gcttcgacaa caggattacg accagctcca tagactccac gaccaacaat gatgatatcg
     6181 ctaccgcagt ttacaatcac ttcttcagga gtacgatatt gctgtcccag cccgtctcct
     6241 ttaacatcca agccgatacc aggggacata gttatgtagt cgctttgaag gttaggaaat
     6301 cgacgaccag ctataaagcc aaagcaaaaa tcggtatgct tctcaaacca ttctaaggtt
     6361 ttctctgtgt aggaaccagt agccaaagag cctttggaag acatttcagc caaaagcaag
     6421 agaccacgtc ccaaaggtaa accaacttct ttgaggcctt gtataatacc ctcgcctggc
     6481 actgtatggc aatttgtgat atgagcccaa gaagcaattt tgtacacacc agatgcatat
     6541 tgtagcttga cggtatttcc aatgtctgcg aatttgcgat cctcaaagat aagaaaacga
     6601 tgctttttac ctaaggccac cagtttttct accatatcct ggtcgaaatc ctcgacaacg
     6661 tcaatatgtg tcttgataac acagacatag ggtccaattt tatctaccaa ttctaagatt
     6721 tcggatttct tcgtcaaatc gaccgcgact gacaagttgc tttgcttttc ttccatcaaa
     6781 gccaacaatt ccttggcaat gggatttttc atcccctcag ctctagctga atagctttga
     6841 aatactctag catccataac tttgctttta aacctttaat ttcgatccaa gcaaaaaaga
     6901 ggttcttggt aggacaatac ggtaagaaaa cacgacatgt gcagagatgc cgacgaagca
     6961 tagttaaact gggatggtaa aatcaattaa gaatttataa agacaaaatt gtataagtct
     7021 ctaaaacatc ttaattatac ctcacagaac tatctaaaat atattcacaa agtgcaaaca
     7081 ttatcatgaa aaagaaccat tttaatttaa agcaagggca ttaaggctta tttacagaat
     7141 ttcttacttt tgtaaagatt ataaggctga ttatcttttt caccatgcca aaaattacac
     7201 aagatagaat ggatgtttga aattaaacgt gagtatacaa acaaatacac taggtaaatc
     7261 gaaacatttt tttctccatt aagtaacaaa ttcctattta gagaaagaat gctgagtaga
     7321 ttaaataatc tatacaaact tttttaacac aaatgcatac atatagccag tgggatttgt
     7381 agctaagctt gcatgcctgc aggtcgatcg actctagagg atcagaaaat tatcgccata
     7441 aaagacagaa taagtcatca gcggttgttt catttcctat attttttttt tattttttta
     7501 ttttttaata agggaaaatt taacgtctaa ggatacagaa gattgttagc acattaaagt
     7561 aataaaggct taagtagtaa gtgccttagc atgttattgt atttcaaagg acataatcta
     7621 aaataataac aatatcattt ctcacaagtt attcaatttt cttttttttt tctaataata
     7681 tcaagaatgt attatttgtt tgacataagt caactaattt atttaatatg ctggattaat
     7741 cttgcagaca tgtaaattaa caagttttag tcaaataacg ttgaagtttc aatgaactca
     7801 aataatttct cttttttttt atataaccat aagtctaatc tgatttatat tttccgcagg
     7861 gatcaactga agttatgaca tttggattgg atcacttata accttggtcg ccaaataata
     7921 caaaaatcag cgttataaaa caaagaaggt ttttgttaag aaattaatcc tctttcttga
     7981 taagaaagtt gaaccgaaat tgcagatact gatatatgaa aataataccc acaattttgg
     8041 gaatagcgca agcctcaatt taaacaatag gtgaggacac atgataatga cctcaatgat
     8101 tgttagaaga aaagagcctc attacaaaat cgaaaaatga atggttgggt acaagtttcc
     8161 aaaacatggt aaagtggact ttgcgtatga gacgtaaata gaaaaaaaca cttgttatat
     8221 gttttctaga attattgttg tctctttatg gttggatgat gcaaaatagt aatttcggtt
     8281 agttgctgta aaacaccacg agacaaatag atatggatat ttattaaatc aggaaaaacg
     8341 taactctcgg ctactggatg gttcagtcac ccaacgatta ctggggagag aaaacagggc
     8401 aaaagcaaag cttaaaggaa tccgattgtc attcggcaat gtgcagcgaa actaaaaacc
     8461 ggataatgga cctgttaatc gaaacattga agatatataa aggaagagga atcctggcat
     8521 atcatcaatt gaataagttg aattaattat ttcaatctca ttctcacttt ctgacttata
     8581 gtcgctttgt taaat
//
