LOCUS Exported 6351 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of human beta B2-crystallin in E. coli. ACCESSION . VERSION . KEYWORDS pE-SUMO CRYBB2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6351) TITLE Larry David lab plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 6351) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6351 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 21..40 /label=T7 /note="T7 promoter, forward primer" promoter 21..39 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 40..64 /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 79..101 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 114..131 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 144..431 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG" CDS 1099..1116 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1169..1187) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1183..1230 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1267..1722 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1354..1373) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1564..1585 /label=F1ori-F /note="F1 origin, forward primer" promoter 1749..1853 /gene="bla" /label=AmpR promoter CDS 1854..2714 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2072..2091) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2885..3473 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3374..3393 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3627..3644 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3659..3801 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3727..3746) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3887..3909 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3903..4094) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4903..5985) /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" primer_bind 5947..5966 /label=LacI-R /note="LacI, reverse primer" promoter complement(5986..6063) /gene="lacI" /label=lacI promoter primer_bind 6269..6288 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc 121 atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca 181 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga 241 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa 301 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg 361 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac 421 agattggagg tgcctcagat caccagaccc aggcgggcaa gccacagtcc ctcaacccca 481 agatcatcat ctttgagcag gaaaactttc aaggccactc gcatgagctc aatgggccct 541 gccccaacct gaaggaaact ggcgtggaga aggcaggttc tgtcctagtg caggctggac 601 cctgggtggg ctatgaacag gccaactgca agggcgagca gtttgtgttt gagaagggtg 661 agtacccccg ctgggactca tggaccagca gccgaaggac ggactccctc agctccctga 721 ggcccatcaa agtggacagc caagagcaca agatcatcct ctatgaaaac cccaacttca 781 ccgggaagaa gatggaaatc atagatgacg atgtacccag cttccacgcc catggctacc 841 aggagaaggt gtcatctgtg cgggtgcaga gtggcacgtg ggttggctac cagtaccccg 901 gctaccgtgg actgcagtac ctgctggaga agggagacta caaggacagc agcgactttg 961 gggcccctca cccccaggtg cagtccgtgc gccgtatccg cgacatgcag tggcaccaac 1021 gtggtgcctt ccacccctcc aactaatcta gaggatccga attcgagctc cgtcgacaag 1081 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 1141 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 1201 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 1261 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 1321 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 1381 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 1441 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 1501 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 1561 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 1621 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 1681 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg 1741 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1801 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 1861 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 1921 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1981 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 2041 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 2101 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 2161 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 2221 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 2281 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 2341 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 2401 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 2461 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 2521 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta 2581 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 2641 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 2701 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 2761 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 2821 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2881 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2941 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 3001 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 3061 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 3121 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 3181 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 3241 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 3301 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 3361 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 3421 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 3481 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 3541 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 3601 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 3661 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac 3721 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 3781 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3841 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3901 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 3961 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 4021 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 4081 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 4141 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 4201 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 4261 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 4321 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 4381 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 4441 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 4501 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 4561 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca 4621 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc 4681 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc 4741 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc 4801 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc 4861 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag 4921 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 4981 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt 5041 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 5101 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt 5161 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc 5221 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 5281 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 5341 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 5401 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 5461 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat 5521 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 5581 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 5641 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 5701 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 5761 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 5821 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 5881 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 5941 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 6001 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 6061 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 6121 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 6181 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc 6241 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg 6301 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc g //