LOCUS Exported 6886 bp ds-DNA circular SYN 03-FEB-2018 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS pTU1-A-pdh_RiboJ_GFP+_MGApt_Bba_B0015_Bmeg SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6886) AUTHORS Karen Polizzi's Lab TITLE Direct Submission JOURNAL Exported Feb 3, 2018 from SnapGene 4.1.3 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6886 /organism="synthetic DNA construct" /mol_type="other DNA" source 450..449 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 1..44 /label=bacterial terminator /note="putative bacterial transcription terminator" terminator 1..44 /label=bacterial terminator /note="putative bacterial transcription terminator" misc_feature 47..66 /label=BioBrick prefix /note="BioBrick prefix for parts that start with ""ATG""" misc_feature 84..646 /label=pdh primer_bind 352..377 /label=SeqpRBBm258_for misc_feature 417..420 /label=T7 promoter /note="T7 promoter" terminator 450..507 /label=his operon terminator /note="This putative transcriptin terminator from the E. coli his operon has a 2-bp deletion introduced during synthesis. Its efficiency has not been determined." misc_RNA 653..702 /label=sTRSV HHRz /note="hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)" primer_bind 756..780 /label=GFP_MGapt_F CDS 760..1497 /codon_start=1 /label=GFPuv3 /translation="MTSSKIWASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERT ISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADK QKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKR DHMVLLEFVTAAGITHGMDEPYK" primer_bind 760..780 /label=GFP_MGapt_F CDS 781..1497 /codon_start=1 /product="Cycle 3 GFP (Crameri et al., 1996)" /label=Cycle 3 GFP /note="mammalian codon-optimized" /translation="ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITHGMDEPYK" primer_bind 1497..1515 /label=Bmeg_MGApt_F /note="Bmeg_MGApt_F; CAGGTCTCTTCGATGAATTCGCGGCCGCATG" misc_feature 1532..1569 /label=MGApt terminator 1578..1649 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 1578..1602 /label=pBP_B0015_Gibson_fwd terminator 1665..1692 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(1691..1717) /label=GFP_MGapt_R primer_bind 1714..1738 /label=Tet_RebB_F primer_bind 1718..1735 /label=VF2 primer_bind complement(1758..1783) /label=PUC_AatII_Inverse_R CDS complement(1862..3238) /codon_start=1 /gene="Bacillus subtilis tetL" /product="tetracycline efflux protein" /label=TcR /note="confers resistance to tetracycline" /translation="MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFN KPPASTNWVNTAFMLTFSIGTAVYGKLSDQLGIKRLLLFGIIINCFGSVIGFVGHSFFS LLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMGEGVGPAIGGMIA HYIHWSYLLLIPMITIITVPFLMKLLKKEVRIKGHFDIKGIILMSVGIVFFMLFTTSYS ISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPY MMKDVHQLSTAEIGSVIIFPGTMSVIIFGYIGGILVDRRGPLYVLNIGVTFLSVSFLTA SFLLETTSWFMTIIIVFVLGGLSFTKTVISTIVSSSLKQQEAGAGMSLLNFTSFLSEGT GIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVYKHSQRDF " CDS complement(1862..3238) /codon_start=1 /gene="Bacillus subtilis tetL" /product="tetracycline efflux protein" /label=TcR /note="confers resistance to tetracycline" /translation="MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFN KPPASTNWVNTAFMLTFSIGTAVYGKLSDQLGIKRLLLFGIIINCFGSVIGFVGHSFFS LLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMGEGVGPAIGGMIA HYIHWSYLLLIPMITIITVPFLMKLLKKEVRIKGHFDIKGIILMSVGIVFFMLFTTSYS ISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPY MMKDVHQLSTAEIGSVIIFPGTMSVIIFGYIGGILVDRRGPLYVLNIGVTFLSVSFLTA SFLLETTSWFMTIIIVFVLGGLSFTKTVISTIVSSSLKQQEAGAGMSLLNFTSFLSEGT GIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVYKHSQRDF " primer_bind 2740..2764 /label=Tet_seq1 CDS complement(3438..4442) /codon_start=1 /gene="repB" /product="RepB replication protein" /label=repB /note="from Enterococcus faecalis plasmid pAM-alpha-1" /db_xref="GI:22652809" /protein_id="AAN03827.1" /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE" CDS complement(3438..4442) /codon_start=1 /gene="repB" /product="RepB replication protein" /label=repB /note="from Enterococcus faecalis plasmid pAM-alpha-1" /db_xref="GI:22652809" /protein_id="AAN03827.1" /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE" primer_bind complement(3462..3481) /label=Tet_seq2 primer_bind 4329..4368 /label=BsaI_X primer_bind complement(4836..4863) /label=Tet_RebB_R misc_feature 4876..4896 /label=BioBrick suffix /note="universal suffix for all parts" misc_feature 4876..4896 /label=BioBrick suffix /note="universal suffix for all parts" primer_bind complement(4954..4973) /label=VR primer_bind complement(5040..5057) /label=Yeastgg_for primer_bind complement(5060..5081) /label=Yeast_for_II rep_origin complement(5073..5661) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" rep_origin complement(5073..5661) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5832..6692) /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" CDS complement(5832..6692) /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(5960..5992) /label=pSB1A2_G239C_RC primer_bind 5960..5992 /label=pSB1A2_G239C promoter complement(6693..6797) /gene="bla" /label=AmpR promoter promoter complement(6693..6797) /gene="bla" /label=AmpR promoter primer_bind 6815..6834 /label=VF2 primer_bind 6817..6837 /label=Tet_RebB_F primer_bind complement(6857..6882) /label=PUC_AatII_Inverse_R ORIGIN 1 atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctggaat tcgcggccgc 61 ttctagaacg tctcaatctc tatgaaggcc tgtgaagttt acaggtcttt tttttggctg 121 aaaaggaagg ttttctgaaa taaagaaaga gtttcgacat gattcacgta aaaattacaa 181 aaggttattg ttttaaaaaa tcgtttattt gtggcaagat aggagatgtg cagacgtaaa 241 aaaagaaaag gttattataa aacatataat acagtttgcg tctgtgtaat agtacagcat 301 gtctttttta tgtgttttat ttcacaaact aaaaaagcgt aatttcttga tgtaaatgca 361 ttgacgaact gttttagtgt attgtaaact aggtagcgta gtaagtgttg tattaatcat 421 ttttgcaaaa taattaatac agtttaacga aacgttcgtt tcgcttaaac tttcctttta 481 cgtaaatgtt tgcacagaat tcgctgggga atacatagaa atgttaagga ttagcgaatt 541 caaaaagcta ctcgacaaaa tatgactttt acttgtccat ttgggacatt ctaaaagcgt 601 gtatgtttgt ttgattagta aaatgaattt atacggctta aatgaagtac agctgtcacc 661 ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca aataattttg 721 tttaaacttg ttcacttaaa tcaaggaggt gaatgtcata tgactagttc gaagatctgg 781 gctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 841 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga 901 aagcttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 961 gtcactactt tgacctatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg 1021 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 1081 aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 1141 aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa 1201 ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga 1261 atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac 1321 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 1381 ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 1441 cttgagtttg taactgctgc tgggattaca catggcatgg atgagcccta caaataatga 1501 attcgcggcc gcatgctcga tatagcaata cggatcccga ctggcgagag ccaggtaacg 1561 aatggatccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 1621 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg 1681 ggcctttctg cgtttatatg ttaccggtcc aagtcgacca cctgacgtct aagaaaccat 1741 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga 1801 attcctgtta taaaaaaagg atcaattttg aactctctcc caaagttgat cccttaacga 1861 tttagaaatc cctttgagaa tgtttatata cattcaaggt aaccagccaa ctaatgacaa 1921 tgattcctga aaaaagtaat aacaaattac tatacagata agttgactga tcaacttcca 1981 taggtaacaa cctttgatca agtaagggta tggataataa accacctaca attgcaatac 2041 ctgttccctc tgataaaaag ctggtaaagt taagcaaact cattccagca ccagcttcct 2101 gctgtttcaa gctacttgaa acaattgttg atataactgt tttggtgaac gaaagcccac 2161 ctaaaacaaa tacgattata attgtcatga accatgatgt tgtttctaaa agaaaggaag 2221 cagttaaaaa gctaacagaa agaaatgtaa ctccgatgtt taacacgtat aaaggacctc 2281 ttctatcaac aagtatccca ccaatgtagc cgaaaataat gacactcatt gttccaggga 2341 aaataattac acttccgatt tcggcagtac ttagctggtg aacatctttc atcatataag 2401 gaaccataga gacaaaccct gctactgttc caaatataat tcccccacaa agaactccaa 2461 tcataaaagg tatatttttc cctaatccgg gatcaacaaa aggatctgtt actttcctga 2521 tatgttttac aaatatcagg aatgacagca cgctaacgat aagaaaagaa atgctatatg 2581 atgttgtaaa caacataaaa aatacaatgc ctacagacat tagtataatt cctttgatat 2641 caaaatgacc ttttatcctt acttctttct ttaataattt cataagaaac ggaacagtga 2701 taattgttat cataggaatg agtagaagat aggaccaatg aatataatgg gctatcattc 2761 caccaatcgc tggaccgact ccttctccca tggctactat cgatccaata agaccaaatg 2821 ctttacccct attttccttt ggaatatagc gcgcaactac aaccattacg agtgctggaa 2881 atgcagctgc accagcccct tgaataaaac gagccataat aagtaaggaa aagaaagaat 2941 ggccaacaaa cccaattacc gacccgaaac aatttattat aattccaaat aggagtaacc 3001 ttttgatgcc taattgatca gatagctttc catatacagc tgttccaatg gaaaaggtta 3061 acataaaggc tgtgttcacc cagtttgtac tcgcaggtgg tttattaaaa tcatttgcaa 3121 tatcaggtaa tgagacgttc aaaaccattt catttaatac gctaaaaaaa gataaaatgc 3181 aaagccaaat taaaatttgg ttgtgtcgta aattcgattg tgaataggat gtattcacat 3241 ttcaccctcc aataatgagg gcagacgtag tttatagggt taatgatacg cttccctctt 3301 ttaattgaac cctgttacat tcattacact tcataattaa ttcctcctaa acttgattaa 3361 aacattttac cacatataaa ctaagtttta aattcagtat ttcatcactt atacaacaat 3421 atggcccgtt tgttgaacta ctctttaata aaataatttt tccgttccca attccacatt 3481 gcaataatag aaaatccatc ttcatcggct ttttcgtcat catctgtatg aatcaaatcg 3541 ccttcttctg tgtcatcaag gtttaatttt ttatgtattt cttttaacaa accaccatag 3601 gagattaacc ttttacggtg taaaccttcc tccaaatcag acaaacgttt caaattcttt 3661 tcttcatcat cggtcataaa atccgtatcc tttacaggat attttgcagt ttcgtcaatt 3721 gccgattgta tatccgattt atatttattt ttcggtcgaa tcatttgaac ttttacattt 3781 ggatcatagt ctaatttcat tgcctttttc caaaattgaa tccattgttt ttgattcacg 3841 tagttttctg tattcttaaa ataagttggt tccacacata ccaatacatg catgtgctga 3901 ttataagaat tatctttatt atttattgtc acttccgttg cacgcataaa accaacaaga 3961 tttttattaa tttttttata ttgcatcatt cggcgaaatc cttgagccat atctgacaaa 4021 ctcttattta attcttcgcc atcataaaca tttttaactg ttaatgtgag aaacaaccaa 4081 cgaactgttg gcttttgttt aataacttca gcaacaacct tttgtgactg aatgccatgt 4141 ttcattgctc tcctccagtt gcacattgga caaagcctgg atttacaaaa ccacactcga 4201 tacaactttc tttcgcctgt ttcacgattt tgtttatact ctaatatttc agcacaatct 4261 tttactcttt cagccttttt aaattcaaga atatgcagaa gttcaaagta atcaacatta 4321 gcgattttct tttctctcca tggtctgact tttccacttt ttgtcttgtc cactaaaacc 4381 cttgattttt catctgaata aatgctacta ttaggacaca taatattaaa agaaaccccc 4441 atctatttag ttatttgttt ggtcacttat aactttaaca gatggggttt ttctgtgcaa 4501 ccaattttaa gggttttcaa tactttaaaa cacatacata ccaacacttc aacgcacctt 4561 tcagcaacta aaataaaaat gacgttattt ctatatgtat caagataaga aagaacaagt 4621 tcaaaaccat caaaaaaaga caccttttca ggtgcttttt ttattttata aactcattcc 4681 ctgatctcga cttcgttctt tttttacctc tcggttatga gttagttcaa attcgttctt 4741 tttaggttct aaatcgtgtt tttcttggaa ttgtgctgtt ttatccttta ccttgtctac 4801 aaacccctta aaaacgtttt taaaggcttt taagccgtct gtacgttcct taaggaaggt 4861 gtttgccaga gacggtacta gtagcggccg ctgcaggctt cctcgctcac tgactcgctg 4921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 4981 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5041 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5101 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5161 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5221 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 5281 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5341 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5401 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5461 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 5521 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 5581 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 5641 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 5701 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 5761 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 5821 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 5881 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 5941 ccatctggcc ccagtgctgc aatgataccg cgagagccac gctcaccggc tccagattta 6001 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 6061 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 6121 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 6181 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 6241 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 6301 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 6361 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 6421 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 6481 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 6541 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 6601 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 6661 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 6721 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 6781 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 6841 attatcatga cattaaccta taaaaatagg cgtatcacga ggcaga //