LOCUS Exported 6051 bp ds-DNA circular SYN 28-JUL-2021 DEFINITION Expresses in bacterial cytoplasm superfolder GFP with SpyTag003 and flanking linkers between Asp102 and Asp103 . ACCESSION . VERSION . KEYWORDS pET28-sfGFP-SpyTag003 Loop B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6051) TITLE DogCatcher allows Loop-friendly Protein-Protein Ligation JOURNAL Unpublished REFERENCE 2 (bases 1 to 6051) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6051 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(778..1860) /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 1822..1841 /label=LacI-R /note="LacI, reverse primer" promoter complement(1861..1938) /gene="lacI" /label=lacI promoter primer_bind 2144..2163 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2247..2266 /label=T7 /note="T7 promoter, forward primer" promoter 2247..2265 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2266..2290 /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 2305..2327 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 3145..3162 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3232..3250) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3246..3293 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3330..3785 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3417..3436) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3627..3648 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3877..4692) /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 4600..4619 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4684..4703 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4814..5402 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5303..5322 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5556..5573 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5588..5727 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5653..5672) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5813..5835 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5829..6020) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg 421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt 481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca 541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag 601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg 661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc 721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac 901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac 961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca 1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc 1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc 1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg 1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca 2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac 2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga 2281 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcgt 2341 aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc 2401 aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg 2461 acgctgaagt tcatctgtac tactggtaaa ctgccggtac cttggccgac tctggtaacg 2521 acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac 2581 ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat 2641 ggcgggggtg gctcgcgtgg tgttccacat atcgttatgg ttgatgcgta caaacgctat 2701 aagggaggag gtggctctga cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc 2761 gataccctgg taaaccgcat tgagctgaaa ggcattgact ttaaagaaga cggcaatatc 2821 ctgggccata agctggaata caattttaac agccacaatg tttacatcac cgccgataaa 2881 caaaaaaatg gcattaaagc gaattttaaa attcgccaca acgtggagga tggcagcgtg 2941 cagctggctg atcactacca gcaaaacact ccaatcggtg atggtcctgt tctgctgcca 3001 gacaatcact atctgagcac gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat 3061 catatggttc tgctggagtt cgtaaccgca gcgggcatca cgcatggtat ggatgaactg 3121 tacaaaggct ccggctccgg ctcccaccat caccatcacc attgataagc tagcaccact 3181 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 3241 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 3301 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc 3361 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3421 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3481 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3541 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 3601 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 3661 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 3721 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac 3781 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 3841 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa 3901 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 3961 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 4021 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 4081 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 4141 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 4201 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 4261 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 4321 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 4381 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 4441 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 4501 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 4561 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 4621 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg 4681 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 4741 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 4801 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 4861 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 4921 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 4981 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 5041 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 5101 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 5161 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 5221 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 5281 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 5341 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 5401 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5461 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5521 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5581 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg 5641 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta 5701 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc 5761 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 5821 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc 5881 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg 5941 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg 6001 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt c //