LOCUS Exported 6072 bp ds-DNA circular SYN 28-JUL-2021 DEFINITION Expresses in bacterial cytoplasm superfolder GFP with DogTag and flanking linkers between Asp102 and Asp103 . ACCESSION . VERSION . KEYWORDS pET28-sfGFP-DogTag Loop B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6072) TITLE DogCatcher allows Loop-friendly Protein-Protein Ligation JOURNAL Unpublished REFERENCE 2 (bases 1 to 6072) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6072 /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 3166..3183 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3253..3271) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3267..3314 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3351..3806 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3438..3457) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3648..3669 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3898..4713) /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 4621..4640 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4705..4724 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4835..5423 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5324..5343 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5577..5594 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5609..5748 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5674..5693) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5834..5856 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5850..6041) /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 ggaggaggtg gctctgatat tccggctaca tacgaattca ccgatggtaa acattatatc 2701 accaatgaac cgataccgcc gaaaggcggg ggtggctcgg acggcacgta caaaacgcgt 2761 gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac 2821 tttaaagaag acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat 2881 gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac 2941 aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt 3001 gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa 3061 gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc 3121 acgcatggta tggatgaact gtacaaaggc tccggctccg gctcccacca tcaccatcac 3181 cattgataag ctagcaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt 3241 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 3301 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg 3361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 3421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 3481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 3541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 3601 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 3661 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 3721 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 3781 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc 3841 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta 3901 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc 3961 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag 4021 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat 4081 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga 4141 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc 4201 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc 4261 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg 4321 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc 4381 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc 4441 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag 4501 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa 4561 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt 4621 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct 4681 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta 4741 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4801 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4861 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4921 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4981 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 5041 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 5101 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 5161 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 5221 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5281 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5341 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5401 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5461 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 5521 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 5581 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt 5641 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 5701 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg 5761 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 5821 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 5881 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc 5941 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata 6001 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg 6061 ggatttctgt tc //