LOCUS Exported 9734 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAVS1 safe harbor targeting vector expressing miniIAA7-3XFlag tagged Seipin. ACCESSION . VERSION . KEYWORDS pSH-EFIRES-P-Seipin-miniIAA7-3XFlag SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9734) AUTHORS Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E TITLE An efficient auxin-inducible degron system with low basal degradation in human cells. JOURNAL Nat Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi: 10.1038/s41592-019-0512-x. PUBMED 31451765 REFERENCE 2 (bases 1 to 9734) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9734 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(135..152) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(306..894) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(386..405) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1065..1925) /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 1688..1707 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1926..2030) /gene="bla" /label=AmpR promoter rep_origin 2057..2512 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2144..2163) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2354..2375 /label=F1ori-F /note="F1 origin, forward primer" misc_feature 3029..3832 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" polyA_signal 3858..3906 /note="synthetic polyadenylation signal" misc_feature 3920..4011 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 3960..3979 /label=RVprimer3 /note="pGL3 vector, forward primer" promoter 4023..5185 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 4254..5176 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 5133..5153 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" intron 5224..5356 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 5339..5358 /label=pMT2-F /note="Synthetic intron, forward primer" promoter 5401..5419 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 6888..6953 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 6991..7543 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7135..7152) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7362..7381 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 7543..8145 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 8042..8062 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 8379..8460 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8416..8435) /label=SV40pA-R /note="SV40 polyA, reverse primer" misc_feature 8488..9324 /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" ORIGIN 1 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta 61 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag 121 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 301 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 901 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 961 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1021 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 1081 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 1141 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 1201 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 1261 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 1321 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 1381 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 1441 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 1501 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 1561 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 1621 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 1681 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 1741 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 1801 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 1861 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 1921 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 1981 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 2041 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2101 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 2281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 2401 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 2461 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttgccattcg 2521 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 2581 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc 2641 gtcgacctac tctcttccgc attggagtcg ctttaactgg ccctggcttt ggcagcctgt 2701 gctgacccat gcagtcctcc ttaccatccc tccctcgact tcccctcttc cgatgttgag 2761 cccctccagc cggtcctgga ctttgtctcc ttccctgccc tgccctctcc tgaacctgag 2821 ccagctccca tagctcagtc tggtctatct gcctggccct ggccattgtc actttgcgct 2881 gccctcctct cgcccccgag tgcccttgct gtgccgccgg aactctgccc tctaacgctg 2941 ccgtctctct cctgagtccg gaccactttg agctctactg gcttctgcgc cgcctctggc 3001 ccactgtttc cccttcccag gcaggtcctg ctttctctga cctgcattct ctcccctggg 3061 cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct ggcccagatc 3121 cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc ttgctctctg 3181 ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc tgcaccacgt 3241 gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct ctcctccctc 3301 acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt ctggggcctg 3361 tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg cgtcccgcct 3421 ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag taccccgtct 3481 ccctggcttt agccacctct ccatcctctt gctttctttg cctggacacc ccgttctcct 3541 gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc cccttacctc 3601 tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg tccgagagct 3661 cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag catgtttgct 3721 gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg gccggttaat 3781 gtggctctgg ttctgggtac ttttatctgt cccctccacc ccacagtggg gcgcatgcgt 3841 ggaattatca cctcgagaat aaaatatctt tattttcatt acatctgtgt gttggttttt 3901 tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac 3961 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tactagtcgt 4021 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 4081 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 4141 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 4201 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 4261 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 4321 ttacttccac ctggctccag tacgtgattc ttgatcccga gctggagcca ggggcgggcc 4381 ttgcgcttta ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg 4441 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct 4501 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt 4561 aaatgcgggc caggatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg 4621 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag 4681 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc 4741 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga 4801 aagatggccg cttcccggcc ctgctccagg gggctcaaaa tggaggacgc ggcgctcggg 4861 agagcgggcg ggtgagtcac ccacacaaag gaaaggggcc tttccgtcct cagccgtcgc 4921 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctggagctt 4981 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac 5041 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt 5101 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt 5161 ttttcttcca tttcaggtgt cgtgaacacg ggctgcagaa gttggtcgtg aggcactggg 5221 caggtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact gggcttgtcg 5281 agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactga catccacttt 5341 gcctttctct ccacaggtgt ccactcccag ttcaattaca gctcttaagg ctagagtact 5401 taatacgact cactataggc tagcctcgag aattcaccat ggtcaacgac cctccagtac 5461 ctgccttact gtgggcccag gaggtgggcc aagtcttggc aggccgtgcc cgcaggctgc 5521 tgctgcagtt tggggtgctc ttctgcacca tcctcctttt gctctgggtg tctgtcttcc 5581 tctatggatc tttttattac tcttacatgc cgacagtcag ccacctcagc cctgtgcatt 5641 tctactacag gaccgactgt gattcctcca ccacctcact ctgctccttc cctgttgcca 5701 atgtctcgct gactaagggt ggacgtgatc gggtgctgat gtatggacag ccgtatcgtg 5761 ttaccttaga gcttgagctg ccagagtccc ctgtgaatca agatttgggc atgttcttgg 5821 tcaccatttc ctgctacacc agaggtggcc gaatcatctc cacttcttcg cgttcggtga 5881 tgctgcatta ccgctcagac ctgctccaga tgctggacac actggtcttc tctagcctcc 5941 tgctatttgg ctttgcagag cagaagcagc tgctggaggt ggaactctac gcagactata 6001 gagagaactc gtacgtgccg accactggag cgatcattga gatccacagc aagcgcatcc 6061 agctgtatgg agcctacctc cgcatccacg cgcacttcac tgggctcaga tacctgctat 6121 acaacttccc gatgacctgc gccttcatag gtgttgccag caacttcacc ttcctcagcg 6181 tcatcgtgct cttcagctac atgcagtggg tgtggggggg catctggccc cgacaccgct 6241 tctctttgca ggttaacatc cgaaaaagag acaattcccg gaaggaagtc caacgaagga 6301 tctctgctca tcagccaggg cctgaaggcc aggaggagtc aactccgcaa tcagatgtta 6361 cagaggatgg tgagagccct gaagatccct cagggacaga gggtcagctg tctgaggagg 6421 agaaaccaga tcagcagccc ctgagcggag aagaggagct agagcctgag gccagtgatg 6481 gttcaggctc ctgggaagat gcagctttgc tgacggaggc caacctgcct gctcctgctc 6541 ctgcttctgc ttctgcccct gtcctagaga ctctgggcag ctctgaacct gctgggggtg 6601 ctctccgaca gcgccccacc tgctctagtt ccggatctgg cggaggaggg agcgggggag 6661 gcggatctgg cggaggcgga tccggcttct ctgagaccgt ggacctgatg ctgaacctgc 6721 agtccaataa ggagggctct gtggatctga agaacgtgag cgccgtgcct aaggagaaga 6781 ccacactgaa ggacccatcc aagccccctg ccaaggcaca ggtggtggga tggccacccg 6841 tgcggaacta cagaaagaat atgatgaccc agcagaagac aagctccgac tacaaagacc 6901 atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac aagtagcggc 6961 cgctctagcc caattccgcc ccccccccta acgttactgg ccgaagccgc ttggaataag 7021 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 7081 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 7141 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 7201 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 7261 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 7321 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 7381 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 7441 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 7501 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatgggcac cgagtacaag 7561 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc 7621 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg 7681 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 7741 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 7801 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 7861 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 7921 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 7981 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 8041 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 8101 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac 8161 ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct ccgaccgaag ccacccgggg 8221 cggccccgcc gaccccgcac ccgcccccga ggcccaccga ctctagctag aggatcataa 8281 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 8341 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 8401 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 8461 attctagttg tggtttgtcc atgcatcact agggacagga ttggtgacag aaaagcccca 8521 tccttaggcc tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag 8581 gcagattcct tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga 8641 acctctaagg tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg 8701 gggtgggagg gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta 8761 ccctctccca gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg 8821 tgctgcagct tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat 8881 aagttggtcc tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt 8941 cctccgtgcg tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg 9001 ctgtggtgag gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta 9061 ggtgttcacc aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt 9121 aaagagtccc cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc 9181 taaggccctg ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag 9241 gcttccctgt cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc 9301 tacaggggtt cctggctctg ctcttcagac tgagccccgt tcccctgcat ccccgttccc 9361 ctgcatcccc cttcccctgc atcccccaga ggccccaggc cacctacttg gcctggaccc 9421 cacgagaggc caccccagcc ctgtctacca ggctgccttt tgggtggatt ctcctccaac 9481 tgtggggtga ctgcttggca aactcactct tcggggtatc ccaggaggcc tggagcattg 9541 gggtgggctg gggttcagag aggagggatt cccttctcag gttacgtggc caagaagcag 9601 gggagctggg tttgggtcag gtctgggtgt ggggtgacca gcttatgctg tttgcccagg 9661 acagcctagt tttagcgctg aaaccctcag tcctaggaaa acagggatgg ttggtcactg 9721 tctctgggct cgac //