LOCUS Exported 9435 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid with 4xMmaPylT cassette and MmaPylRS for amber suppression and incorporation of acetyl-lysine; for transient or stable piggyBac-mediated integration. ACCESSION . VERSION . KEYWORDS pAS_4xMma PylT_FLAG-AcKRS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9435) TITLE Elsaesser amber suppression piggyBac vectors REFERENCE 2 (bases 1 to 9435) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9435 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(1..105) /gene="bla" /label=AmpR promoter rep_origin complement(131..586) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(268..289) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 480..499 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 713..735 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 727..744 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 728..744 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 874..936 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter 2727..2938 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 2951..3219 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 3269..3278 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3278..3301 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 4685..5271 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4852..4869) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5079..5098 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5284..5883 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5284..5303) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5780..5800 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(5943..5963) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 6005..6126 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6042..6061) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6096..6115 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 6889..6923 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(7414..7430) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7414..7430) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7427..7449) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7438..7454 /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(7462..7492) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7507..7528 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7645..7662) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7816..8404) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7896..7915) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8575..9435) /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 9198..9217 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1321 cacctggggg gatacgggga aaaggcctcc acggccaact agtgaaaaac cgcacttgtc 1381 cggaaacccc gggaatctaa cccggctgaa cggatttaga gtccgttcga tctacatgat 1441 caggtttccg aggtacccag gcggcgcaca agctatataa acctgaagga aatctcaact 1501 ttacacttag gtcaagttac ttatcgtact agagcttcag caggaaattt aactaaaatc 1561 taatttaacc agcatagcaa atatcattta ttcccaaaat gctaaagttt gagataaacg 1621 gacttgattt ccggctgttt tgacactatc cagaatgcct tgcagatggg tggggcatgc 1681 taaatactgc aggaaaaacc gcacttgtcc ggaaaccccg ggaatctaac ccggctgaac 1741 ggatttagag tccgttcgat ctacatgatc aggtttccga ggtacccagg cggcgcacaa 1801 gctatataaa cctgaaggaa atctcaactt tacacttagg tcaagttact tatcgtacta 1861 gagcttcagc aggaaattta actaaaatct aatttaacca gcatagcaaa tatcatttat 1921 tcccaaaatg ctaaagtttg agataaacgg acttgatttc cggctgtttt gacactatcc 1981 agaatgcctt gcagatgggt ggggcatgct aaatactgca ggaaaaaccg cacttgtccg 2041 gaaaccccgg gaatctaacc cggctgaacg gatttagagt ccgttcgatc tacatgatca 2101 ggtttccgag gtacccaggc ggcgcacaag ctatataaac ctgaaggaaa tctcaacttt 2161 acacttaggt caagttactt atcgtactag agcttcagca ggaaatttaa ctaaaatcta 2221 atttaaccag catagcaaat atcatttatt cccaaaatgc taaagtttga gataaacgga 2281 cttgatttcc ggctgttttg acactatcca gaatgccttg cagatgggtg gggcatgcta 2341 aatactgcag gaaaaaccgc acttgtccgg aaaccccggg aatctaaccc ggctgaacgg 2401 atttagagtc cgttcgatct acatgatcag gtttccgagg tacccaggcg gcgcacaagc 2461 tatataaacc tgaaggaaat ctcaacttta cacttaggtc aagttactta tcgtactaga 2521 gcttcagcag gaaatttaac taaaatctaa tttaaccagc atagcaaata tcatttattc 2581 ccaaaatgct aaagtttgag ataaacggac ttgatttccg gctgttttga cactatccag 2641 aatgccttgc agatgggtgg ggcatgctaa atactgcaga actagtgctc tagaaaggat 2701 ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 2761 gttgggggga ggggtcggca attgaacggg tgcctagaga aggtggcgcg gggtaaactg 2821 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 2881 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagct 2941 gaagcttcga ggggctcgca tctctccttc acgcgcccgc cgccctacct gaggccgcca 3001 tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc 3061 cgccgtctag gtaagtttaa agctcaggtc gagaccgggc ctttgtccgg cgctcccttg 3121 gagcctacct agactcagcc ggctctccac gctttgcctg accctgcttg ctcaactcta 3181 cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc aagctgtgac cggcgcctac 3241 tctagagcta gcgtttaaac ttaagcttgc caccatggac tacaaggacg acgacgacaa 3301 gatggacaag aagcccctga acaccctgat cagcgccaca ggactgtgga tgtccagaac 3361 cggcaccatc cacaagatca agcaccacga ggtgtcccgg tccaaaatct acatcgagat 3421 ggcctgcggc gatcacctgg tcgtcaacaa cagcagaagc agccggacag ccagagccct 3481 gcggcaccac aagtacagaa agacctgcaa gcggtgcaga gtgtccgacg aggacctgaa 3541 caagttcctg accaaggcca acgaggacca gaccagcgtg aaagtgaagg tggtgtccgc 3601 ccccacccgg accaagaaag ccatgcccaa gagcgtggcc agagccccca agcccctgga 3661 aaacaccgaa gccgctcagg cccagcccag cggcagcaag ttcagccccg ccatccccgt 3721 gtctacccag gaaagcgtca gcgtccccgc cagcgtgtcc accagcatct ctagcatctc 3781 aaccggcgcc acagcttctg ccctggtcaa gggcaacacc aaccccatca ccagcatgtc 3841 tgcccctgtg caggcctctg ccccagccct gaccaagtcc cagaccgacc ggctggaagt 3901 gctcctgaac cccaaggacg agatcagcct gaacagcggc aagcccttcc gggagctgga 3961 aagcgagctg ctgagccggc ggaagaagga cctccagcaa atctacgccg aggaacggga 4021 gaactacctg ggcaagctgg aaagagagat cacccggttc ttcgtggacc ggggcttcct 4081 ggaaatcaag agccccatcc tgatccccct ggagtacatc gagcggatgg gcatcgacaa 4141 cgacaccgag ctgagcaagc agattttccg ggtggacaag aacttctgcc tgcggcccat 4201 gatggccccc aacatcttca actacgcccg gaaactggat cgcgctctgc ccgaccccat 4261 caagattttc gagatcggcc cctgctaccg gaaagagagc gacggcaaag agcacctgga 4321 agagtttaca atgctgaact ttttccagat gggcagcggc tgcaccagag agaacctgga 4381 atccatcatc accgactttc tgaaccacct ggggatcgac ttcaagatcg tgggcgacag 4441 ctgcatggtg tacggcgaca ccctggacgt gatgcacggc gacctggaac tgtctagcgc 4501 cgtcgtggga cccatccctc tggaccggga gtggggcatc gataagccct ggatcggagc 4561 cggcttcggc ctggaacggc tgctgaaagt caagcacgac tttaagaaca tcaagcgggc 4621 tgccagaagc gagagctact acaacggcat cagcaccaac ctgtgataag gatccgcggc 4681 cgcgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc 4741 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 4801 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 4861 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 4921 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 4981 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 5041 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 5101 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 5161 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc 5221 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggcgtcc 5281 ggaatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc 5341 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg 5401 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 5461 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 5521 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 5581 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag 5641 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 5701 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc 5761 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 5821 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 5881 taggtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac 5941 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgcgtta 6001 actaaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 6061 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 6121 atcttatcat gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg 6181 gtctcccttc cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac 6241 tgggttctta tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga 6301 acgaagtccc taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt 6361 ttttagggcc cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa 6421 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 6481 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 6541 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 6601 ggggggctgt ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag 6661 tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag 6721 ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc 6781 gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca 6841 gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt 6901 ttacgcagac tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt 6961 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc 7021 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct 7081 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc 7141 acgcctttaa cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt 7201 tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg 7261 acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt 7321 acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg 7381 agttacccgg cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 7441 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 7501 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 7561 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 7621 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 7681 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 7741 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 7801 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 7861 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 7921 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 7981 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 8041 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 8101 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 8161 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 8221 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 8281 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 8341 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 8401 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 8461 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 8521 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 8581 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 8641 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 8701 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 8761 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 8821 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 8881 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 8941 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 9001 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 9061 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 9121 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 9181 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 9241 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 9301 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 9361 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 9421 atgttgaata ctcat //