LOCUS Exported 9288 bp ds-DNA circular SYN 23-JUL-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9288) AUTHORS Chen TITLE Direct Submission JOURNAL Exported Jul 24, 2018 from SnapGene Viewer 4.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9288 /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" misc_feature 874..936 /label=3' TR misc_feature 1113..1344 /label=Core Insulator misc_feature 1346..1366 /label=H1 misc_feature 1367..1977 /label=SH LHA misc_feature 1886..1888 /label=PAM misc_feature 1889..1907 /label=SH gRNA 4 misc_feature 1960..1968 /label=SHS231 misc_feature 1978..2030 /label=Bxb1 attP promoter 2066..3244 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2297..3235 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" protein_bind 3256..3289 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 3298..4029 /codon_start=1 /product="photostable variant of TagRFP (Shaner et al., 2008)" /label=TagRFP-T /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV ARYCDLPSKLGHKLNGMDELYK" CDS 4036..4089 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4093..5124 /codon_start=1 /label=HygromycinB Resistance /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK DLQ" misc_feature 5132..5173 /label=fC31 attP misc_feature 5174..5701 /label=SH RHA misc_feature 5352..5354 /label=PAM misc_feature 5355..5373 /label=SH gRNA 5 misc_feature 5702..5722 /label=H2 polyA_signal 5863..5980 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" misc_feature complement(6237..6467) /label=Core Insulator misc_feature 6742..6776 /label=5' TR primer_bind complement(7267..7283) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 7291..7307 /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(7315..7345) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7360..7381 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(7669..8257) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8428..9288) /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" 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 ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca 1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca 1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 1321 acacctgggg ggatacgggg aaaaggcctc caaggccaga ctagaggtac ccattgagtt 1381 ctgaggtggt ttgtcataca gcaatggcaa tcagatatat ccacacaaat ataattttag 1441 tttatatttc tgctactgca gttctcatct tattctgagg atatgtgaca aaataattct 1501 ttcaaaaata ttgatgctgt gctagattac tattttgaat gaattactag acaaatactt 1561 catatgtatc tttttttttt tttttttttt tttttttttt tttttttgag acggagtctc 1621 gctctgtcgc ccaggctgga gtgcagtggc gggatctcgg ctcactgcaa tatgtatctt 1681 attatgtggg tttacacatt atttatctta ttgatttaac ttcaaaacta aactttagtt 1741 tagctcttgg gccctatctg gaaagggtca tcttttaatc accattaaat cactgaagtc 1801 atcagtttat tcaaagtact ctgcacaaat tagcattctt tagtggttgt gaaataaata 1861 gactttaaac ttatcattaa tattcccaat ggtactatgg gggaggcaaa attttctgtc 1921 ttcttagtgg tttttttttt ttggctaggg ctaaggatta aactgtcaag cttcgaggtg 1981 gtttgtctgg tcaaccaccg cggtctcagt ggtgtacggt acaaaccccg agtcgacctt 2041 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc 2101 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag 2161 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg 2221 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt 2281 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg 2341 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc 2401 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc 2461 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg 2521 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg 2581 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca 2641 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac 2701 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca 2761 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc 2821 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc 2881 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc 2941 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta 3001 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg 3061 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt 3121 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc 3181 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc 3241 gtgaggaatt agcttataac ttcgtatagc atacattata cgaagttatc ctgcaggatg 3301 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc 3361 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc 3421 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc 3481 ctggctacca gcttcatgta cggcagcaga accttcatca accacaccca gggcatcccc 3541 gatttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa 3601 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac 3661 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca 3721 ctcggctggg aggccaacac cgagatgctg taccccgctg acggcggcct ggaaggcaga 3781 accgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca 3841 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggaccac 3901 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct 3961 gtggccagat actgcgacct ccctagcaaa ctggggcaca aacttaatgg catggacgag 4021 ctgtacaagg ctagcgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 4081 cctggcccaa ccatgaaaaa gcctgaactc accgctacct ctgtcgagaa gtttctgatc 4141 gaaaagttcg acagcgtctc cgacctgatg cagctctccg agggcgaaga atctcgggct 4201 ttcagcttcg atgtgggagg gcgtggatat gtcctgcggg tgaatagctg cgccgatggt 4261 ttctacaaag atcgctatgt ttatcggcac tttgcatccg ccgctctccc tattcccgaa 4321 gtgcttgaca ttggggagtt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag 4381 ggtgtcacct tgcaagacct gcctgaaacc gaactgcccg ctgttctcca gcccgtcgcc 4441 gaggccatgg atgccatcgc tgccgccgat cttagccaga ccagcgggtt cggcccattc 4501 ggacctcaag gaatcggtca atacactaca tggcgcgatt tcatctgcgc tattgctgat 4561 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcctc cgtcgcccag 4621 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac 4681 gccgatttcg gctccaacaa tgtcctgacc gacaatggcc gcataacagc cgtcattgac 4741 tggagcgagg ccatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg 4801 ccctggttgg cttgtatgga gcagcagacc cgctacttcg agcggaggca tcccgagctt 4861 gcaggatctc ctcggctccg ggcttatatg ctccgcattg gtcttgacca actctatcag 4921 agcttggttg acggcaattt cgatgatgca gcttgggctc agggtcgctg cgacgcaatc 4981 gtccggtccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgctgccgtc 5041 tggaccgatg gctgtgtgga agtgctcgcc gatagtggaa acagacgccc cagcactcgt 5101 cctagggcaa aggatctgca gtaatctaga agtgccccaa ctggggtaac ctttgagttc 5161 tctcagttgg gggactagtg cgtggacaaa gagcagtaaa ttgtgaaaat ggaacaaggc 5221 aagggggctt agactacagt agttaatcat caagaagtga caaaaaaaat aatggttagt 5281 taataagatt tgtttaagca gatttctccc agctttagct ctctgtctct ggtgatcaga 5341 atgcactcct tccttcagac tcagtgagca catcttccac atggaagatt tcttctctag 5401 cttttaggaa atccagagaa ccctttttgt atctgctgtt tttttttttt taaatgtctt 5461 taactcaaaa caatttatgt gccaggatga catatctttg gataatgtgt tctgaactcc 5521 ttcagtacat acgtatataa attaaagcaa atatttttta tgataagctg gcataatagt 5581 ttcataattt aatcactgat ttaaaaattt aattaaaatt attttttgat attttgtgta 5641 ataatttttg aggagtatct tttgtgctta atgagtggca gatgacaccc atgttcttag 5701 ctacgcgtta actaagtaag cggccgcccc ggtatcgaca atcaacctct ggattacaaa 5761 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 5821 gctgctttaa tgcctttgta tcatgcgtta actaactaaa cttgtttatt gcagcttata 5881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 5941 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg aattgactca 6001 aatgatgtca attagtctat cagaagctat ctggtctccc ttccggggga caagacatcc 6061 ctgtttaata tttaaacagc agtgttccca aactgggttc ttatatccct tgctctggtc 6121 aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt ccctaaagaa acagtggcag 6181 ccaggtttag ccccggaatt gactggattc cttttttagg gcccattggt atggcttttt 6241 ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag 6301 cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg 6361 cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg 6421 gagggacgta attacatccc tgggggcttt gggggggggc tgtccctgat atctataaca 6481 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat 6541 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc 6601 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc 6661 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga 6721 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa 6781 tctagctgca tcaggatcat atcgtcgggt cttttttccg gctcagtcat cgcccaagct 6841 ggcgctatct gggcatcggg gaggaagaag cccgtgcctt ttcccgcgag gttgaagcgg 6901 catggaaaga gtttgccgag gatgactgct gctgcattga cgttgagcga aaacgcacgt 6961 ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt taacggtgaa ctgttcgttc 7021 aggccacctg ggataccagt tcgtcgcggc ttttccggac acagttccgg atggtcagcc 7081 cgaagcgcat cagcaacccg aacaataccg gcgacagccg gaactgccgt gccggtgtgc 7141 agattaatga cagcggtgcg gcgctgggat attacgtcag cgaggacggg tatcctggct 7201 ggatgccgca gaaatggaca tggatacccc gtgagttacc cggcgggcgc gcttggcgta 7261 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 7321 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 7381 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 7441 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 7501 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 7561 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 7621 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 7681 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 7741 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 7801 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 7861 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 7921 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 7981 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 8041 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 8101 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 8161 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 8221 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 8281 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 8341 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 8401 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 8461 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 8521 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 8581 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 8641 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 8701 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 8761 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 8821 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 8881 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 8941 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 9001 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 9061 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 9121 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 9181 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 9241 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcat //