LOCUS Exported 7076 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION protein expression in E. coli. ACCESSION . VERSION . KEYWORDS pET30a-FluB-NP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7076) AUTHORS Shen H, Forgacs D, Chapla D, Moremen KW, Wells L, Hamer SA, Tompkins SM, Ross TM, Rouphael N, Edupuganti S, Collins MH, Tarleton RL. TITLE A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses. JOURNAL medRxiv 2021.(pre-print) 01.20.21249279 REFERENCE 2 (bases 1 to 7076) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7076 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 969..988 /label=LacI-R /note="LacI, reverse primer" promoter complement(1008..1085) /gene="lacI" /label=lacI promoter primer_bind 1291..1310 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1398..1417 /label=T7 /note="T7 promoter, forward primer" promoter 1398..1416 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1417..1441 /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)." RBS 1456..1478 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1489..1506 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1516..1533 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1540..1584 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 1600..1614 /codon_start=1 /product="enterokinase recognition and cleavage site" /label=enterokinase site /translation="DDDDK" CDS 3100..3111 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 3334..3351 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3404..3422) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3418..3465 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3502..3957 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3589..3608) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3799..3820 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4049..4864) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 4772..4791 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4856..4875 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4986..5574 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5475..5494 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5728..5745 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5760..5899 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5825..5844) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5985..6007 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6001..6192) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7001..1007) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 181 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg 241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg 1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc 1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag 1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga 1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc 1441 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca ccatcatcat 1501 catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 1561 ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 1621 gctgatatcg gatccatgag caacatggac attgatggta tgaacaccgg caccattgac 1681 aagaccccgg aggaaatcac cagcggtacc agcggtacca cccgtccgat cattcgtccg 1741 gcgaccctgg cgccgccgag caacaagcgt acccgtaacc cgagcccgga gcgtgcgacc 1801 accagcagcg aagacgatgt tggtcgtaaa gcgcagaaga aacaaacccc gaccgagatc 1861 aagaaaagcg tgtacaacat ggtggttaag ctgggtgaat tctataacca gatgatggtt 1921 aaagcgggcc tgaacgacga tatggagcgt aacctgattc aaaacgcgca cgcggtggaa 1981 cgtatcctgc tggcggcgac cgacgataag aaaaccgagt ttcagaagaa aaagaacgcg 2041 cgtgacgtga aagaaggcaa ggaggaaatt gatcacaaca agaccggtgg caccttctac 2101 aagatggttc gtgacgataa aaccatctat tttagcccga tccgtattac cttcctgaaa 2161 gaggaagtga agaccatgta caaaaccacc atgggcagcg acggttttag cggcctgaac 2221 cacatcatga ttggccacag ccagatgaac gacgtgtgct tccaacgtag caaagcgctg 2281 aagcgtgttg gtctggaccc gagcctgatt agcacctttg cgggcagcac cgttccgcgt 2341 cgtagcggtg cgaccggtgt ggcgatcaaa ggtggcggta ccctggttgc ggaagcgatt 2401 cgtttcatcg gtcgtgcgat ggcggaccgt ggcctgctgc gtgatattaa agcgaagacc 2461 gcgtatgaga agatcctgct gaacctgaaa aacaagtgca gcgcgccgca gcaaaaagcg 2521 ctggttgatc aggtgatcgg tagccgtaac ccgggcattg cggacatcga agatctgacc 2581 ctgctggcgc gtagcatggt ggttgtgcgt ccgagcgttg cgagcaaggt tgtgctgccg 2641 atcagcattt acgcgaaaat tccgcagctg ggttttaacg ttgaggaata cagcatggtg 2701 ggctatgagg cgatggcgct gtataacatg gcgaccccgg ttagcatcct gcgtatgggt 2761 gacgatgcga aagacaagag ccaactgttc tttatgagct gcttcggcgc ggcgtacgag 2821 gatctgcgtg tgctgagcgc gctgaccggt accgaattta aaccgcgtag cgcgctgaaa 2881 tgcaagggct tccacgttcc ggcgaaggag caggtggaag gcatgggtgc ggcgctgatg 2941 agcattaaac tgcagttttg ggcgccgatg acccgtagcg gtggtaacga ggcgggtggt 3001 gacggtggta gcggtcagat tagctgcagc ccggttttcg cggtggaacg tccgatcgcg 3061 ctgagcaagc aagcggtgcg tcgtatgctg agcatgaaca tcgagggtcg tgacgcggat 3121 gttaagggca acctgctgaa aatgatgaac gacagcatgg cgaaaaagac cagcggtaac 3181 gcgtttattg gcaaaaagat gttccagatc agcgataaaa acaagaccaa cccgatcgag 3241 attccgatca aacaaaccat cccgaacttc tttttcggtc gtgacaccgc ggaagattat 3301 gacgatctgg ataagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg 3361 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 3421 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 3481 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 3541 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 3601 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 3661 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 3721 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 3781 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 3841 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 3901 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg 3961 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 4021 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact 4081 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg 4141 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga 4201 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat 4261 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca 4321 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat 4381 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt 4441 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat 4501 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg 4561 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg 4621 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg 4681 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc 4741 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat 4801 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc 4861 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa 4921 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 4981 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 5041 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 5101 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 5161 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 5221 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 5281 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 5341 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 5401 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 5461 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 5521 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 5581 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 5641 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 5701 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 5761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc 5821 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg 5881 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 5941 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 6001 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 6061 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 6121 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 6181 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc 6241 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact 6301 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac 6361 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca 6421 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag 6481 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 6541 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 6601 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg 6661 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 6721 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 6781 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 6841 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 6901 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 6961 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 7021 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcg //