LOCUS       sequence-12968-e       10332 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 10332)
  AUTHORS   Zhang H, Fire AZ
  TITLE     Cell autonomous specification of temporal identity by Caenorhabditis
            elegans microRNA lin-4.
  JOURNAL   Dev Biol. 2010 Aug 15. 344(2):603-10.
  PUBMED    20493184
REFERENCE   2  (bases 1 to 10332)
  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: "Dev Biol. 
            2010 Aug 15. 344(2):603-10."
COMMENT     Sequence Label: pPD133.47
FEATURES             Location/Qualifiers
     source          1..10332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2493..2513
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2556..2584)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3332..3352
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3485..3507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7906..7928)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8028..8047
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(8125..8143)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8211..8315
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8316..8384,8385..9176)
                     /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(8534..8553)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9347..9935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9836..9855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10089..10106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10223..10244
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10259..10264,10265..10282,10283..10289)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10297..10313
                     /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     10302..10324
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(10321..10332,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(10321..10332,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag cttgggctgc aggtcggcta taataagttc ttgaataaaa
       61 taattttccc gacaaaacat gagtatttct ttcgaaaata aaagtgcagg ctaattagag
      121 attattctgt aattaactgc ataatttgtc acgtgccata gttttacatt ccactacgtc
      181 atagttctta aaatactaat ctcctgaaaa tagaagtagg tgaagaaagt ttaattatca
      241 gttctaaaat gacaattgat ctttggaata tgttctgaaa ctaccgatca ttgaacagat
      301 gctatttgaa tgatatagaa ttgtatattt gcaatttctg aaacgcgttc ttaaaggcac
      361 acagattaat tcaaaagggt ctggccgcaa aaaggtttat ggtggccgat tttgagtttt
      421 gtgtgtgatt gctttttcac aatcagtgtt ttcaggatta tgtgatgaac tagatcttca
      481 agtttcgtta catttcatat gttttcggaa ctcacgaagt acatattggg tattgtgctc
      541 aaaaaattca gcaatcagct tcgctccgct gactttagaa cccaaaaaaa tagtatggcc
      601 aaactgactg tgttacgatc atttcaattt ttcaatacat atttaagatt tctaagagta
      661 agaaggtcaa aaactgttct ggaatacata tatatttttc aggttacaat tagtcaaaaa
      721 gtgcactgaa atatacgttt taatttcacg aataacccaa ttagttcaat gtatttttgg
      781 tcaaccaacg ttaaagtttg gcttccaacc aattatcatt tctgatcaac cacaatgttt
      841 tttctttatc tgcaagttaa ttttttattt ttatccagat gtttggcata tttttcaatt
      901 cttcactagc gcccacttct tgcacttccg gcgccctgaa tctaatgcat ctgttgcaag
      961 aattgaaaga ccaatcaaca cattgttttc ttcacgagat actgaagaaa atgaataaaa
     1021 acagagaaaa agagccatgt gattagtgac aactgttgct aacagatacc atagcttgga
     1081 cttggtacgt gatggcaacg tatgggtcaa caaaaatgat tgcagagggg gtgcaaaaca
     1141 gtcaagtcga gaaaatatga aaaacagaaa acaaagaaca gaaaaatggg tttgagagtc
     1201 agtataattt ataaaagaaa aattgtacat agaaattaac catttttgta gaagaagtta
     1261 tttttcaagc atcgttaaaa attattcaaa gcaccttatt tcatatttaa ttttaaacat
     1321 ggttaaatga acaacacggt gcgcaatcag gaaaacttga aatctgaaac tgttgttgtg
     1381 atcttcttcg caactgttca gatagcacta gtgtaatgtt aagagtgcgc gaatataatg
     1441 gaatataatg gatcacacct cctgccatca ggtaaacgtc tctgttatca catatttcca
     1501 actattaaat ttttaccttt tacagtttta catttttttg aaaaaagtaa ctttttgtct
     1561 tcaaaatccc tgacgaaaat atcaaatatt ttaatcgaga ctgcagagga accgattgat
     1621 gatttggaaa atccagcttt acctgtgtaa gaactgaaaa gtttcataac cctagggtat
     1681 tcccagttac attccccact ggctaacaat agcacccagt ttttcatcac ctttcttcaa
     1741 atttctcggc gatttgttaa aaacaaaatt tgtgtccctt ctctgatatc tctatgtctc
     1801 taaacacaag ttcatcggaa aacgaaggag ggtaggtgtt ggttgggctc ccgaagtgaa
     1861 aatagaagag caagaataga atattagaga gagagtgcag agagggcggg atagctcccg
     1921 ggattccgtt ttcttcttct ttatcttcaa cgatgatgtg tgtgcgtgtt gtatagattc
     1981 tgttgctccc ccacaactcg ctccgaaggc tcaatacaat tcaattgata ttggaggaga
     2041 gcctaccgga gtgggaggat aagaagaaac ataagaagaa gaagaagaag aagcatgctt
     2101 ctggtttttg atgctatgaa aacggcacaa aaagatgatt gaggtccctt ttcaatacct
     2161 tctctcatct ttcaaatccc attgaaacct aaaacttctc accacgcttt accattgttc
     2221 tccaaaaact tatagcaatg tctataactt ttttatctct gaaaagcagt gttccatttt
     2281 tctttttcct attttatttc aattgtttct cacatttcgt ttggattctt tgcttgtcaa
     2341 ccagcttctt cttccacttt taccgtctaa ttttcagggc agggagccat caaacccacg
     2401 accactagat ccatctagag gatccccggg attggccaaa ggacccaaag gaggaccctt
     2461 ggagggtacc gagctcagaa aaaatgactg ctccaaagaa gaagcgtaag gtaccggtag
     2521 aaaaaatgag taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
     2581 atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat
     2641 acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt ccatgggtaa
     2701 gtttaaacat atatatacta actaaccctg attatttaaa ttttcagcca acacttgtca
     2761 ctactttctc atggggcgtt caatgcttct cgagataccc agatcatatg aaacagcatg
     2821 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
     2881 atgacgggaa ctacaagaca cgtaagttta aacagttcgg tactaactaa ccatacatat
     2941 ttaaattttc aggtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
     3001 taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact
     3061 atatttcaca caacgtatac atcactgccg acaaacaaaa gaatgggatc aaagcggtaa
     3121 gtttaaacat gattttacta actaactaat ctgatttaaa ttttcagaac ttcaaaatta
     3181 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa
     3241 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt
     3301 cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg
     3361 ggattacaca tggcatggat gaactataca aatgcccggt gggtgaagac cagaaacagc
     3421 acctcgaact gagccgcgat attgcccagc gtttcaacgc gctgtatggc gagatcgatc
     3481 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3541 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3601 cccaacagtt gcgaaggtaa gtttaaacag atccatacta actaacttgt tctgacataa
     3661 ttttcagctt gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg
     3721 aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc
     3781 agatgcacgg ttacgatgcg cccatctaca ccaacgtaac ctatcccatt acggtcaatc
     3841 cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg
     3901 aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttaac tcggcgtttc
     3961 atctgtggtg caacgggcgc tgggtcggtt acggccaggt aagtttaatt aagttgatac
     4021 taactaacaa agatctgatt aattttcagg acagtcgttt gccgtctgaa tttgacctga
     4081 gcgcattttt acgcgccgga gaaaaccgcc tcgcggtgat ggtgctgcgt tggagtgacg
     4141 gcagttatct ggaagatcag gatatgtggc ggatgagcgg cattttccgt gacgtctcgt
     4201 tgctgcataa accgactaca caaatcagcg atttccatgt tgccactcgc tttaatgatg
     4261 atttctcccg cgctgtactg gaggctgaag tccaggtaag tttaaacagg atcttactaa
     4321 ctaacatgct aacactgaat tttcagatgt gcggcgagtt gcgtgactac ctacgggtaa
     4381 cagtttcttt atggcagggt gaaacgcagg tcgccagcgg caccgcgcct ttcggcggtg
     4441 aaattatcga tgagcgtggt ggttatgccg atcgcgtcac actacgtctg aacgtcgaaa
     4501 acccgaaact gtggagcgcc gaaatcccga atctctatcg tgcggtggtt gaactgcaca
     4561 ccgccgacgg cacgctgatt gaagcagaag cctgcgatgt cggtttccgc gaggtgcgga
     4621 ttgaaaatgg tctgctgctg ctgaacggca agccgttgct gattcgaggc gttaaccgtc
     4681 acgagcatca tcctctgcat ggtcaggtca tggatgagca gacgatggtg caggatgtaa
     4741 gtttaaacta ttcgttacta actaacttta aacatttaaa ttttcagatc ctgctgatga
     4801 agcagaacaa ctttaacgcc gtgcgctgtt cgcattatcc gaaccatccg ctgtggtaca
     4861 cgctgtgcga ccgctacggc ctgtatgtgg tggatgaagc caatattgaa acccacggca
     4921 tggtgccaat gaatcgtctg accgatgatc cgcgctggct accggcgatg agcgaacgcg
     4981 taacgcgaat ggtgcagcgc gatcgtaatc acccgagtgt gatcatctgg tcgctgggga
     5041 atggtaagtt taaacagttg aatactaact aacggagatc tttgaaattt tcagaatcag
     5101 gccacggcgc taatcacgac gcgctgtatc gctggatcaa atctgtcgat ccttcccgcc
     5161 cggtgcagta tgaaggcggc ggagccgaca ccacggccac cgatattatt tgcccgatgt
     5221 acgcgcgcgt ggatgaagac cagcccttcc cggctgtgcc gaaatggtcc atcaaaaaat
     5281 ggctttcgct acctggagag acgcgcccgc tgattctttg cgaggtaagt ttaaacagaa
     5341 ctctactaac taacacatta gatcctaatt ttcagtacgc tcacgcgatg ggcaacagtc
     5401 ttggcggttt cgctaaatac tggcaggcgt ttcgtcagta tccccgttta cagggcggct
     5461 tcgtctggga ctgggtggat cagtcgctga ttaaatatga tgaaaacggc aacccgtggt
     5521 cggcttacgg cggtgatttt ggcgatacgc cgaacgatcg ccagttctgt atgaacggtc
     5581 tggtctttgc cgaccgcacg ccgcatccag cgtaagttta aacaataacc tactaactaa
     5641 cgtagataat ttaaattttc aggctgacgg aagcaaaaca ccagcagcag tttttccagt
     5701 tccgtttatc cgggcaaacc atcgaagtga ccagcgaata cctgttccgt catagcgata
     5761 acgagctcct gcactggatg gtggcgctgg atggtaagcc gctggcaagc ggtgaagtgc
     5821 ctctggatgt cgctccacaa ggtaaacagt tgattgaact gcctgaacta ccgcagccgg
     5881 agagcgccgg gcaactctgg ctcacagtac gcgtagtgca accgaacgcg accgcatggt
     5941 cagaagccgg gcacatcagc gcatggcagc agtggaggta agtttaaaca agatcctact
     6001 aactaactct acattgatga attttcagac tggcggaaaa cctcagtgtg acgctccccg
     6061 ccgcgtccca cgccatcccg catctgacca ccagcgaaat ggatttttgc atcgagctgg
     6121 gtaataagcg ttggcaattt aaccgccagt caggctttct ttcacagatg tggattggcg
     6181 ataaaaaaca actgctgacg ccgctgcgcg atcagttcac ccgtgcaccg ctggataacg
     6241 acattggcgt aagtgaagcg acccgcattg accctaacgc ctgggtcggt aagtttaaac
     6301 aaagttgtac taactaacga agatcttgat aattttcaga acgctggaag gcggcgggcc
     6361 attaccaggc cgaagcagcg ttgttgcagt gcacggcaga tacacttgct gatgcggtgc
     6421 tgattacgac cgctcacgcg tggcagcatc aggggaaaac cttatttatc agccggaaaa
     6481 cctaccggat tgatggtagt ggtcaaatgg cgattaccgt tgatgttgaa gtggcgagcg
     6541 atacaccgca tccggcgcgg attggcctga actgccagct ggcgcaggta gcagagcggg
     6601 taaactggct cggattaggg ccgcaagaaa actatcccga ccgccttact gccgcctgtt
     6661 ttgaccgctg ggatctgcca ttgtcagaca tgtagtaagt ttaaacttga tagtactaac
     6721 taacatgttt catttaaatt ttcagtaccc cgtacgtctt cccgagcgaa aacggtctgc
     6781 gctgcgggac gcgcgaattg aattatggcc cacaccagtg gcgcggcgac ttccagttca
     6841 acatcagccg ctacagtcaa cagcaactga tggaaaccag ccatcgccat ctgctgcacg
     6901 cggaagaagg cacatggctg aatatcgacg gtttccatat ggggattggt ggcgacgact
     6961 cctggagccc gtcagtatcg gcggaattcc aactgagcgc cggtcgctac cattaccaac
     7021 ttgtctggtg tcaaaaataa taggggccgc tgtcatcaga gtaagtttaa actgagttct
     7081 actaactaac gagtaatatt taaattttca gcatctcgcg cccgtgcctc tgacttctaa
     7141 gtccaattac tcttcaacat ccctacatgc tctttctccc tgtgctccca ccccctattt
     7201 ttgttattat caaaaaaact tcttcttaat ttctttgttt tttagcttct tttaagtcac
     7261 ctctaacaat gaaattgtgt agattcaaaa atagaattaa ttcgtaataa aaagtcgaaa
     7321 aaaattgtgc tccctccccc cattaataat aattctatcc caaaatctac acaatgttct
     7381 gtgtacactt cttatgtttt ttttacttct gataaatttt ttttgaaaca tcatagaaaa
     7441 aaccgcacac aaaatacctt atcatatgtt acgtttcagt ttatgaccgc aatttttatt
     7501 tcttcgcacg tctgggcctc tcatgacgtc aaatcatgct catcgtgaaa aagttttgga
     7561 gtatttttgg aatttttcaa tcaagtgaaa gtttatgaaa ttaattttcc tgcttttgct
     7621 ttttgggggt ttcccctatt gtttgtcaag agtttcgagg acggcgtttt tcttgctaaa
     7681 atcacaagta ttgatgagca cgatgcaaga aagatcggaa gaaggtttgg gtttgaggct
     7741 cagtggaagg tgagtagaag ttgataattt gaaagtggag tagtgtctat ggggtttttg
     7801 ccttaaatga cagaatacat tcccaatata ccaaacataa ctgtttccta ctagtcggcc
     7861 gtacgggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     7921 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     7981 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
     8041 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     8101 ataccgcatc aggcggcctt aagggcctcg tgatacgcct atttttatag gttaatgtca
     8161 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     8221 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     8281 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     8341 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     8401 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     8461 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     8521 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     8581 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     8641 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     8701 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     8761 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     8821 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     8881 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     8941 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     9001 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     9061 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     9121 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     9181 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     9241 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     9301 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     9361 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     9421 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     9481 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     9541 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     9601 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     9661 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     9721 gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     9781 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     9841 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     9901 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     9961 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
    10021 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
    10081 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
    10141 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
    10201 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
    10261 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
    10321 caggaaacag ct
//
