LOCUS Exported 7634 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a granzyme B FRET reporter. ACCESSION . VERSION . KEYWORDS pLNCX2-CFP-VGPDFGR-YFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7634) AUTHORS Choi PJ, Mitchison TJ TITLE Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells. JOURNAL Proc Natl Acad Sci U S A. 2013 Apr 16;110(16):6488-93. doi: 10.1073/pnas.1221312110. Epub 2013 Apr 1. PUBMED 23576740 REFERENCE 2 (bases 1 to 7634) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7634 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 414..589 /label=5' LTR (truncated) /note="truncated long terminal repeat from Moloney murine sarcoma virus" misc_feature 652..851 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 1052..1468 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1398..1420 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1436..1452 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" CDS 1512..2306 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1566..1585) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2176..2195 /label=Neo-F /note="Neomycin resistance gene, forward primer" enhancer 2378..2681 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2682..2885 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2836..2856 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 2948..2957 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2954..3670 /codon_start=1 /product="enhanced CFP" /label=ECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3734..4453 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" LTR 4536..5129 /note="long terminal repeat from Moloney murine leukemia virus" primer_bind complement(5232..5254) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 5340..5480 /label=bom /note="basis of mobility region from pBR322" primer_bind 5395..5414 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(5495..5512) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5666..6254) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5746..5765) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6425..7285) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7048..7067 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7286..7390) /gene="bla" /label=AmpR promoter primer_bind 7458..7476 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat 61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc 121 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca 181 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 241 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa 301 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac 361 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa 421 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac 481 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg 541 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt 601 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc 661 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg 721 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt 781 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg 841 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt 901 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa 961 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct 1021 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt 1081 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa 1141 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc 1201 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc 1261 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt 1321 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc 1381 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta 1441 tccagccctc actccttctc taggcgccgg aattccgatc tgatcaagag acaggatgag 1501 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 1561 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 1621 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 1681 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 1741 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 1801 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 1861 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 1921 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 1981 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 2041 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 2101 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 2161 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 2221 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 2281 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgtaatag taatcaatta 2341 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 2401 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 2461 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 2521 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 2581 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 2641 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 2701 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 2761 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 2821 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 2881 gagctggttt agtgaaccgt cagatccgct agcgctaccg gactcagatc agctagcgct 2941 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 3001 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 3061 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 3121 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 3181 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 3241 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 3301 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 3361 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 3421 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 3481 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 3541 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 3601 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 3661 gctgtacaag tccggactca gatctagcgg cgtgggcccc gacttcggcc gcgtgtacgg 3721 cagcggatcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 3781 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 3841 cgatgccacc tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt 3901 gccctggccc accctcgtga ccaccctggg ctacggcctg cagtgcttcg cccgctaccc 3961 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 4021 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 4081 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 4141 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga 4201 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg 4261 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 4321 gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg 4381 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 4441 gctgtacaag taagaattca aggcctctcg acaggcctta atggcctaac atcgataaaa 4501 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt 4561 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata 4621 gagaagttca gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata 4681 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat 4741 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat 4801 ggtccccaga tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg 4861 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct 4921 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact 4981 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct 5041 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat 5101 tgactacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg gagacccctg 5161 cccagggacc accgacccac caccgggagg taagctggct gcctcgcgcg tttcggtgat 5221 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 5281 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 5341 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat 5401 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 5461 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 5581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 5641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 5701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 5761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 5821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 5881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 5941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 6121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 6361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 6421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 6481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 6541 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 6601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 6721 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 6781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7021 gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag 7081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 7441 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattcatac 7501 cagatcaccg aaaactgtcc tccaaatgtg tccccctcac actcccaaat tcgcgggctt 7561 ctgcctctta gaccactcta ccctattccc cacactcacc ggagccaaag ccgcggccct 7621 tccgtttctt tgct //