LOCUS pWHERE. 9944 bp DNA circular SYN 06-APR-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS pWHERE. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9944) AUTHORS caoheibi TITLE Direct Submission REFERENCE 2 (bases 1 to 9944) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..9944 /mol_type="other DNA" /organism="synthetic DNA construct" misc_feature complement(1..2032) /label=H19 ins /note="H19 ins" /note="mH19 insulators on either side of the lacZ transcription unit. Both insulators are expected to protect the integrated transcriptional LacZ unit from negative as well as positive influences from neighboring sequences. Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or by their ability to protect adjacent transgenes from position effects. The fragment of the differential methylated region (DMD) located between the mouse Igf2 and H19 acts as a powerful insulator. The enhancer blocking activity of the DMD fragment is dependent upon four responsive elements to the vertebrate enhancer-blocking protein CTCF. Two mouse DMD fragments have been introduced in opposite orientation in the pWHERE plasmid to insulate your promoter of interest cloned upstream of the new CpG-free LacZ gene from the 5' and 3' adjacent regions at the integrated site in transgenic mice." CDS 2302..2322 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" polyA_signal 5170..5742 /label=EF-1-alpha poly(A) signal /note="human EF-1-alpha polyadenylation signal" misc_feature complement(5750..7781) /label=H19 ins /note="H19 ins" /note="mH19 insulators on either side of the lacZ transcription unit. Both insulators are expected to protect the integrated transcriptional LacZ unit from negative as well as positive influences from neighboring sequences. Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or by their ability to protect adjacent transgenes from position effects. The fragment of the differential methylated region (DMD) located between the mouse Igf2 and H19 acts as a powerful insulator. The enhancer blocking activity of the DMD fragment is dependent upon four responsive elements to the vertebrate enhancer-blocking protein CTCF. Two mouse DMD fragments have been introduced in opposite orientation in the pWHERE plasmid to insulate your promoter of interest cloned upstream of the new CpG-free LacZ gene from the 5' and 3' adjacent regions at the integrated site in transgenic mice." rep_origin complement(7877..8465) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8639..9496) /label=AmpR /note="beta-lactamase" promoter complement(9497..9601) /label=AmpR promoter ORIGIN 1 cagctttgtc acagcggacc ccaacctatg ccgcgtctgc cgagcaatat gtagtattgt 61 actgccacca cgcggcatcg tctgtccatt tagctatagc caaatctgca cagcgtggag 121 agtgaaccga tcgatcgtga agggcgcaag actgaaggag ctacccaaga aatgtgtgtt 181 gtaccacccc atgaccctta tgaatcattg aatgctatgc ctgagtgacc catgagtttg 241 ccataggtga accgcaattt tggtcacctc tatgataaag tatgtgggac tgcactggtc 301 gctgctcggc aacttcggtc ttaccagcca ctgacgatct cgggctgtgt agggaatgag 361 tcaagttctc tggttcagtg tgtaagggaa ccattccaga ggtgcacaca tcttaccacc 421 cctatgaatc cctatttggg tgaccctggg atattgctgg gaatgaatcg ctccccccaa 481 gttggcagca tttgggccac gatatatagg agtatgctgc caccgcgcgg tagcatccgt 541 tcccttgttg cacataacag cttctatgcc ttcctatagt gagccacact ggctggtttt 601 ggggttcagt gaccaaaggg accccctcca gaaacacaag tgttccaacc tcttataaac 661 catatcgacc actgaggcat agcggcttcg gacattgctg tgggcgaacc cgaactttgg 721 cccttggaca ttgtcatggg caaaccttaa ctttggctgt ctctggaaca taatgtttaa 781 gatgacagtc accagcgcag caatttggtc tttccactca caacggcttt tgtgctttct 841 ggcatcgaac cacatgcact ggtttatggg gtctgagacc aaaggagacc attccggaag 901 ggcataggtg tcctgccttc tgcttttaac aaggctctcc gggacagtgc aaaacaggtg 961 aaccccaact ttgccataag tacgattatc tgccacaaac cagccagggg tcctaccacc 1021 tcttcaattg attttgggct gacacccaag gcttgatgta ggattcctca gctgccaagc 1081 tggcagctga cccccattga gagagaatgc agtttcagaa ttgtttgcag ccctgagccg 1141 gagatcatta gcatctgaac gccccaatta gaatacgaaa gcgcaaatca ccagactttc 1201 ttgttggcgg ttccttaagt gattctttgg gtagggaggt caggtgtcct catagatgtg 1261 cagaatttga ggaccatgct tagtggggtc tgcattatgg tactgcaata cattccatga 1321 tcaccacaca tagtagctat acttcaattt tcacacaata gcgctgatgg ccccagaacc 1381 ctataagtca gatacctgag atagctcttg agaacgtttt atcaaggact agcatgaacc 1441 cctggcctca tgaagcccat gactatggga tcatagatgg tgatagggga gaaaactcaa 1501 tcagttgcaa tccgttttag gactgcgatg tacgagactt cactgccgcc gtgcggcaac 1561 cctggtcttt acacacaaag gattctttgc agagagtaag ccgaccttgt tgatttggga 1621 gtccgagtcc acgaggtacc agcctagaaa atgcatgtgt cctgcccccc tagtggcatt 1681 tgtgaccccc ctgaggtact gaacttgggt gacccacagc attgccattt gtgaattcca 1741 ataccagggg tgggggggct ctttaggttt ggcgcaatcg attttgctgc caccacgcgg 1801 caactcccgc gtataaaccc cacaactgat tcagcagacg tccaagaata gggcatggtc 1861 tccttgcaga attcttatgc ctcctggatg ctcgtgtgaa tgtagcatgt tcctttgagt 1921 cctgggtgta agtgccctgc acgctcatcc cccggacatg aaaatagaat ctctattttc 1981 tcaccaacct ttttctttcc ttgtggtgat tcgggaactg taggcaatgg ctcctgcagg 2041 tcctaggtgg atcctctcga gtcccgggcc atggaccctg ttgtgctgca aaggagagac 2101 tgggagaacc ctggagtgac ccagctcaac agactggctg cccaccctcc ctttgcctct 2161 tggaggaact ctgaggaagc caggacagac aggcccagcc agcagctcag gtctctcaat 2221 ggagagtgga ggtttgcctg gttccctgcc cctgaagctg tgcctgagtc ttggctggag 2281 tgtgacctcc cagaggcagt tcccaagaag aagaggaaag ttgaggctga cactgttgtg 2341 gtgccaagca actggcagat gcatggctat gatgccccca tctacaccaa tgtcacctac 2401 cccatcactg tgaacccccc ttttgtgccc actgagaacc ccactggctg ctacagcctg 2461 accttcaatg ttgatgagag ctggctgcaa gaaggccaga ccaggatcat ctttgatgga 2521 gtcaactctg ccttccacct ctggtgcaat ggcaggtggg ttggctatgg ccaagacagc 2581 aggctgccct ctgagtttga cctctctgcc ttcctcagag ctggagagaa caggctggct 2641 gtcatggtgc tcaggtggtc tgatggcagc tacctggaag accaagacat gtggaggatg 2701 tctggcatct tcagggatgt gagcctgctg cacaagccca ccacccagat ttctgacttc 2761 catgttgcca ccaggttcaa tgatgacttc agcagagctg tgctggaggc tgaggtgcag 2821 atgtgtggag aactcagaga ctacctgaga gtcacagtga gcctctggca aggtgagacc 2881 caggtggcct ctggcacagc cccctttgga ggagagatca ttgatgagag aggaggctat 2941 gctgacagag tcaccctgag gctcaatgtg gagaacccca agctgtggtc tgctgagatc 3001 cccaacctct acagggctgt tgtggagctg cacactgctg atggcaccct gattgaagct 3061 gaagcctgtg atgttggatt cagagaagtc aggattgaga atggcctgct gctgctcaat 3121 ggcaagcctc tgctcatcag gggagtcaac aggcatgagc accaccctct gcatggacaa 3181 gtgatggatg aacagacaat ggtgcaagat atcctgctaa tgaagcagaa caacttcaat 3241 gctgtcaggt gctctcacta ccccaaccac cctctctggt acaccctgtg tgacaggtat 3301 ggcctgtatg ttgttgatga agccaacatt gagacacatg gcatggtgcc catgaacagg 3361 ctcacagatg accccaggtg gctgcctgcc atgtctgaga gagtgaccag gatggtgcag 3421 agagacagga accacccctc tgtgatcatc tggtctctgg gcaatgagtc tggacatgga 3481 gccaaccatg atgctctcta caggtggatc aagtctgttg accccagcag acctgtgcag 3541 tatgaaggag gtggagcaga caccacagcc acagacatca tctgccccat gtatgccagg 3601 gttgatgagg accagccctt ccctgctgtg cccaagtgga gcatcaagaa gtggctctct 3661 ctgcctggag agaccagacc tctgatcctg tgtgaatatg cacatgcaat gggcaactct 3721 ctgggaggct ttgccaagta ctggcaagcc ttcagacagt accccaggct gcaaggagga 3781 tttgtgtggg actgggtgga ccaatctctc atcaagtatg atgagaatgg caacccctgg 3841 tctgcctatg gaggagactt tggtgacacc cccaatgaca ggcagttctg catgaatggc 3901 ctggtctttg cagacaggac ccctcaccct gccctcacag aggccaagca ccagcaacag 3961 ttcttccagt tcaggctgtc tggacagacc attgaggtga catctgagta cctcttcagg 4021 cactctgaca atgagctcct gcactggatg gtggccctgg atggcaagcc tctggcttct 4081 ggtgaggtgc ctctggatgt ggcccctcaa ggaaagcagc tgattgaact gcctgagctg 4141 cctcagccag agtctgctgg acaactgtgg ctaacagtga gggtggttca gcccaatgca 4201 acagcttggt ctgaggcagg ccacatctct gcatggcagc agtggaggct ggctgagaac 4261 ctctctgtga ccctgcctgc tgcctctcat gccatccctc acctgacaac atctgaaatg 4321 gacttctgca ttgagctggg caacaagaga tggcagttca acaggcagtc tggcttcctg 4381 tctcagatgt ggattggaga caagaagcag ctcctcaccc ctctcaggga ccaattcacc 4441 agggctcctc tggacaatga cattggagtg tctgaggcca ccaggattga cccaaatgct 4501 tgggtggaga ggtggaaggc tgctggacac taccaggctg aggctgccct gctccagtgc 4561 acagcagaca ccctggctga tgctgttctg atcaccacag cccatgcttg gcagcaccaa 4621 ggcaagaccc tgttcatcag cagaaagacc tacaggattg atggctctgg acagatggca 4681 atcacagtgg atgtggaggt tgcctctgac acacctcacc ctgcaaggat tggcctgaac 4741 tgtcaactgg cacaggtggc tgagagggtg aactggctgg gcttaggccc tcaggagaac 4801 taccctgaca ggctgacagc tgcctgcttt gacaggtggg acctgcctct gtctgacatg 4861 tacacccctt atgtgttccc ttctgagaat ggcctgaggt gtggcaccag ggagctgaac 4921 tatggtcctc accagtggag gggagacttc cagttcaaca tctccaggta ctctcagcaa 4981 cagctcatgg aaacctctca caggcacctg ctccatgcag aggagggaac ctggctgaac 5041 attgatggct tccacatggg cattggagga gatgactctt ggtctccttc tgtgtctgct 5101 gagttccagt tatctgctgg caggtaccac tatcagctgg tgtggtgcca gaagtaaacc 5161 tgagctagca ttatccctaa tacctgccac cccactctta atcagtggtg gaagaacggt 5221 ctcagaactg tttgtttcaa ttggccattt aagtttagta gtaaaagact ggttaatgat 5281 aacaatgcat cgtaaaacct tcagaaggaa aggagaatgt tttgtggacc actttggttt 5341 tcttttttgc gtgtggcagt tttaagttat tagtttttaa aatcagtact ttttaatgga 5401 aacaacttga ccaaaaattt gtcacagaat tttgagaccc attaaaaaag ttaaatgaga 5461 aacctgtgtg ttcctttggt caacaccgag acatttaggt gaaagacatc taattctggt 5521 tttacgaatc tggaaacttc ttgaaaatgt aattcttgag ttaacacttc tgggtggaga 5581 atagggttgt tttcccccca cataattgga aggggaagga atatcattta aagctatggg 5641 agggtttctt tgattacaac actggagaga aatgcagcat gttgctgatt gcctgtcact 5701 aaaacaggcc aaaaactgag tccttgggtt gcatagaaag ctgcctgcag agctttgtca 5761 cagcggaccc caacctatgc cgcgtctgcc gagcaatatg tagtattgta ctgccaccac 5821 gcggcatcgt ctgtccattt agctatagcc aaatctgcac agcgtggaga gtgaaccgat 5881 cgatcgtgaa gggcgcaaga ctgaaggagc tacccaagaa atgtgtgttg taccacccca 5941 tgacccttat gaatcattga atgctatgcc tgagtgaccc atgagtttgc cataggtgaa 6001 ccgcaatttt ggtcacctct atgataaagt atgtgggact gcactggtcg ctgctcggca 6061 acttcggtct taccagccac tgacgatctc gggctgtgta gggaatgagt caagttctct 6121 ggttcagtgt gtaagggaac cattccagag gtgcacacat cttaccaccc ctatgaatcc 6181 ctatttgggt gaccctggga tattgctggg aatgaatcgc tccccccaag ttggcagcat 6241 ttgggccacg atatatagga gtatgctgcc accgcgcggt agcatccgtt cccttgttgc 6301 acataacagc ttctatgcct tcctatagtg agccacactg gctggttttg gggttcagtg 6361 accaaaggga ccccctccag aaacacaagt gttccaacct cttataaacc atatcgacca 6421 ctgaggcata gcggcttcgg acattgctgt gggcgaaccc gaactttggc ccttggacat 6481 tgtcatgggc aaaccttaac tttggctgtc tctggaacat aatgtttaag atgacagtca 6541 ccagcgcagc aatttggtct ttccactcac aacggctttt gtgctttctg gcatcgaacc 6601 acatgcactg gtttatgggg tctgagacca aaggagacca ttccggaagg gcataggtgt 6661 cctgccttct gcttttaaca aggctctccg ggacagtgca aaacaggtga accccaactt 6721 tgccataagt acgattatct gccacaaacc agccaggggt cctaccacct cttcaattga 6781 ttttgggctg acacccaagg cttgatgtag gattcctcag ctgccaagct ggcagctgac 6841 ccccattgag agagaatgca gtttcagaat tgtttgcagc cctgagccgg agatcattag 6901 catctgaacg ccccaattag aatacgaaag cgcaaatcac cagactttct tgttggcggt 6961 tccttaagtg attctttggg tagggaggtc aggtgtcctc atagatgtgc agaatttgag 7021 gaccatgctt agtggggtct gcattatggt actgcaatac attccatgat caccacacat 7081 agtagctata cttcaatttt cacacaatag cgctgatggc cccagaaccc tataagtcag 7141 atacctgaga tagctcttga gaacgtttta tcaaggacta gcatgaaccc ctggcctcat 7201 gaagcccatg actatgggat catagatggt gataggggag aaaactcaat cagttgcaat 7261 ccgttttagg actgcgatgt acgagacttc actgccgccg tgcggcaacc ctggtcttta 7321 cacacaaagg attctttgca gagagtaagc cgaccttgtt gatttgggag tccgagtcca 7381 cgaggtacca gcctagaaaa tgcatgtgtc ctgcccccct agtggcattt gtgacccccc 7441 tgaggtactg aacttgggtg acccacagca ttgccatttg tgaattccaa taccaggggt 7501 gggggggctc tttaggtttg gcgcaatcga ttttgctgcc accacgcggc aactcccgcg 7561 tataaacccc acaactgatt cagcagacgt ccaagaatag ggcatggtct ccttgcagaa 7621 ttcttatgcc tcctggatgc tcgtgtgaat gtagcatgtt cctttgagtc ctgggtgtaa 7681 gtgccctgca cgctcatccc ccggacatga aaatagaatc tctattttct caccaacctt 7741 tttctttcct tgtggtgatt cgggaactgt aggcaatggc tttaattaaa aacccgcttc 7801 ggcgggtttt tttatgcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 7861 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 7921 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 7981 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 8041 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 8101 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 8161 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 8221 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 8281 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 8341 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 8401 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 8461 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 8521 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 8581 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 8641 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 8701 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 8761 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 8821 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 8881 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 8941 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 9001 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 9061 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 9121 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 9181 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 9241 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 9301 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 9361 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 9421 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 9481 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 9541 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 9601 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 9661 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg 9721 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 9781 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta 9841 actatgcggc atcagagcag attgtactga gagtgcacca tatggatctc gataacaaaa 9901 aaccccgccc cggcggggtt ttttgttagc ggccgcttaa ttaa //