LOCUS Exported 5496 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of GFP nano body. ACCESSION . VERSION . KEYWORDS pOPINE GFP nanobody SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5496) AUTHORS Kubala MH, Kovtun O, Alexandrov K, Collins BM TITLE Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. JOURNAL Protein Sci. 2010 Dec;19(12):2389-401. doi: 10.1002/pro.519. PUBMED 20945358 REFERENCE 2 (bases 1 to 5496) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5496 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 310..587 /label=chicken beta-actin promoter primer_bind 866..888 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 950..969 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind 1011..1030 /label=T7 /note="T7 promoter, forward primer" promoter 1011..1029 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1030..1054 /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)." promoter 1070..1179 /label=p10 promoter /note="baculovirus promoter for expression in insect cells" CDS 1200..1541 /codon_start=1 /product="GFP-binding fragment of a single-chain camelid antibody (Rothbauer et al., 2008)" /label=GFP nanobody /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW GQGTQVTVSS" CDS 1545..1562 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1652..1671) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 1717..1772 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(1771..1790) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 1860..1907 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_recomb 1920..2625 /label=baculovirus recombination region (ORF1629) /note="contains part of ORF1629" 3'UTR 2008..2380 /label=polyhedrin 3' UTR /note="3' UTR from the Autographa californica nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)" CDS complement(2037..2625) /codon_start=2 /product="baculovirus capsid-associated protein" /label=ORF1629 /note="required for viral replication" /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA" protein_bind 2634..2667 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(2835..3422) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2914..2933) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3435..4295) /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 4058..4077 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" misc_recomb 4538..5365 /label=baculovirus recombination region (lef2/ORF603) /note="contains ORF603 and part of lef2" CDS 4538..4662 /codon_start=3 /product="baculovirus late expression factor 2" /label=lef2 /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY" CDS complement(4700..5305) /codon_start=1 /product="baculovirus ORF603 protein" /label=ORF603 /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catgcatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 361 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 421 gggggggggg gcgcgcgccg gggggggggg gggggggsgg ggggsggggs ggggsgaggc 481 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 541 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc 601 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 661 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctccttcg ggctgtaatt 721 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc 781 tccgggaggg ccctttgtgc ggggggagcg gctcggggct gtccgcgggg ggacggctgc 841 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga 901 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg 961 gttattgtgc tgtctcatca ttttggcaaa gaattggatc ggaccgaaat taatacgact 1021 cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg acctttaatt 1081 caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt tgtatattaa 1141 ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat accatgcagg 1201 ttcaactggt ggaaagcggc ggtgctctgg tacaaccggg cggtagtctg cgcctgagct 1261 gtgccgcaag cggtttccca gtcaaccgct actctatgcg ttggtatcgc caggcgcctg 1321 gtaaagaacg tgaatgggtt gccggcatga gcagtgcggg cgatcgttct agttacgagg 1381 actctgttaa aggtcgtttt acaattagcc gtgatgatgc gcgcaatacc gtgtatctgc 1441 aaatgaacag tctgaagccg gaggacaccg cagtatatta ttgcaatgtc aacgtggggt 1501 ttgaatattg gggccagggg actcaggtga cggtgagctc taaacatcac catcaccatc 1561 actaagtgat taacctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa 1621 tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg 1681 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg 1741 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc 1801 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac 1861 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgcg 1921 gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa aggagtttgc accagacgca 1981 cctctgttca ctggtccggc gtattaaaac acgatacatt gttattagta catttattaa 2041 gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca 2101 ttaaatttat aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct 2161 ttatatctga atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt 2221 tcaaacaagg gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac 2281 gccacaaaac ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt 2341 ttgttttgta ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca 2401 tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt taaatagagc ttggacatat 2461 ttaacatcgg gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg 2521 ttagaagttg cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg 2581 gctaacacgt ccgcgatcaa atttgtagtt gagctttttg gaattgcgat cgcataactt 2641 cgtatagcat acattatacg aagttataag ctcggaacgc tgcgctcggt cgttcggctg 2701 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 2761 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2821 gcgttgctgg cgtttttcca taggctccgc cccctgacga gcatcacaaa aatcgacgct 2881 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2941 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3001 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 3061 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3121 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3181 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3241 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 3301 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3361 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 3421 aagaagatcc tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3481 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3541 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3601 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3661 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3721 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3781 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3841 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3901 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3961 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 4021 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 4081 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 4141 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 4201 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4261 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 4321 agcatttatc agggttattg tctcatgtcc gcgcgtttcc tgcatctttt aatcaaatcc 4381 caagatgtgt ataaacgcgc cggtatgtac aggaagaggt ttatactaaa ctgttacatt 4441 gcaaacgtgg tttcgtgtgc caagtgtgaa aaccgatgtt taatcaaggc tctgacgcat 4501 ttctacaacc acgactccaa gtgtgtgggt gaagtcatgc atcttttaat caaatcccaa 4561 gatgtgtata aaccaccaaa ctgccaaaaa atgaaaactg tcgacaagct ctgtccgttt 4621 gctggcaact gcaagggtct caatcctatt tgtaattatt gaataataaa acaattataa 4681 atgtcaaatt tgttttttat taacgataca aaccaaacgc aacaagaaca tttgtagtat 4741 tatctataat tgaaaacgcg tagttataat cgctgaggta atatttaaaa tcattttcaa 4801 atgattcaca gttaatttgc gacaatataa ttttattttc acataaacta gacgccttgt 4861 cgtcttcttc ttcgtattcc ttctcttttt catttttctc ttcataaaaa ttaacatagt 4921 tattatcgta tccatatatg tatctatcgt atagagtaaa ttttttgttg tcataaatat 4981 atatgtcttt tttaatgggg tgtatagtac cgctgcgcat agtttttctg taatttacaa 5041 cagtgctatt ttctggtagt tcttcggagt gtgttgcttt aattattaaa tttatataat 5101 caatgaattt gggatcgtcg gttttgtaca atatgttgcc ggcatagtac gcagcttctt 5161 ctagttcaat tacaccattt tttagcagca ccggattaac ataactttcc aaaatgttgt 5221 acgaaccgtt aaacaaaaac agttcacctc ccttttctat actattgtct gcgagcagtt 5281 gtttgttgtt aaaaataaca gccattgtaa tgagacgcac aaactaatat cacaaactgg 5341 aaatgtctat caatatatag ttgctgatgg ccggccgtaa tgagacgcac aaactaatat 5401 cacaaactgg aaatgtctat caatatatag ttgctctagt tattaatagt aatcaattac 5461 ggggtcatta gttcatagcc catatatgga gttccg //