LOCUS Exported 8871 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral vector to express PKA KTR mClover under PGK promoter (With Puromycin Resistance). ACCESSION . VERSION . KEYWORDS pLentiPGK Puro DEST PKAKTRClover SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8871) AUTHORS Regot S, Hughey JJ, Bajar BT, Carrasco S, Covert MW TITLE High-sensitivity measurements of multiple kinase activities in live single cells. JOURNAL Cell. 2014 Jun 19;157(7):1724-34. doi: 10.1016/j.cell.2014.04.039. PUBMED 24949979 REFERENCE 2 (bases 1 to 8871) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8871 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 27..131 /gene="bla" /label=AmpR promoter CDS 132..992 /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 complement(350..369) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1163..1751 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1652..1671 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1905..1922 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2039..2060 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2075..2105 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2113..2129 /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)." primer_bind 2118..2140 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2137..2153 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2137..2153 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2172..2192 /label=T3 /note="T3 promoter, forward primer" promoter 2174..2192 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" LTR 2448..2628 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 2672..2797 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 3290..3523 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 3708..3752 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 4019..4136 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 4183..4693 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 4612..4630 /label=hPGK-F /note="Human PGK promoter, forward primer" protein_bind 4748..4772 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(5033..5054) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 5641..5662 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(5698..5722) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 5753..6341 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5806..5826) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6224..6235) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" promoter 6355..6854 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(6390..6413) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 6787..6806 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 6875..7474 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6875..6894) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7371..7391 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 7612..7845 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 7917..8038 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7954..7973) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8008..8027 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 8057..8192 /label=SV40 ori /note="SV40 origin of replication" primer_bind 8119..8138 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(8224..8243) /label=T7 /note="T7 promoter, forward primer" promoter complement(8225..8243) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(8253..8270) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(8253..8269) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8262..8284) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 8414..8869 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8501..8520) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8711..8732 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2161 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 2221 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 2281 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 2341 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 2401 gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacggg tctctctggt 2461 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 2521 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 2581 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 2641 cagggacctg aaagcgaaag ggaaaccaga gctctctcga cgcaggactc ggcttgctga 2701 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag 2761 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag 2821 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca 2881 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac 2941 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga 3001 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga 3061 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac 3121 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt 3181 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca 3241 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt 3301 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg acgctgacgg 3361 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta 3421 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa 3481 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct 3541 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc 3601 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca 3661 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag 3721 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc 3781 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt 3841 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga 3901 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag 3961 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cggttaactt 4021 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag 4081 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttatcg 4141 atcacgagac tagcctcgag aagcttgata tcgaattccc acggggttgg ggttgcgcct 4201 tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa 4261 acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg 4321 gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg 4381 ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac 4441 tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc 4501 tgtggccaat agcggctgct cagcggggcg cgccgagagc agcggccggg aaggggcggt 4561 gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc 4621 attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga 4681 cctctctccc cagggggatc caccggttcg tcgactagtc cagtgtggtg gaattctgca 4741 gatatcaaca agtttgtaca aaaaagcagg ctccaccatg gagatgccag aggaacccgc 4801 caacagtggg cattcactac ccccggttta tatttacagt cccgagtatg tcagcatctg 4861 cgactccctt gtgaaggtcc ccaaacgggc cagtatggtc aacgaagacg aggcgccatc 4921 tagaaggaag gcgtcgggtc aggtgtcgtc aaggctggag cgcctgaccc tccagtccag 4981 cactagtatg gtgagcaagg gcgaggagct gttcaccggg gttgtgccca tcctggtcga 5041 gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg agggcgatgc 5101 caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 5161 gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct accccgacca 5221 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 5281 catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt tcgagggcga 5341 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 5401 ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca 5461 gaagaacggc atcaaggcta acttcaagat ccgccacaac gttgaggacg gcagcgtgca 5521 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 5581 caaccactac ctgagccatc agtccgccct gagcaaagac cccaacgaga agcgcgatca 5641 catggtcctg ctggagttcg tgaccgccgc ctaagcggcc gcactcgaga tatctagacc 5701 cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctcg acaatcaacc 5761 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 5821 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 5881 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 5941 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 6001 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 6061 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 6121 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt 6181 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 6241 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 6301 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggaattc taccgggtag 6361 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt 6421 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac 6481 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt 6541 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca 6601 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca 6661 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt 6721 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag 6781 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat 6841 ctccgggcct ttcgacctgc agcccaagct taccatgacc gagtacaagc ccacggtgcg 6901 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga 6961 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct 7021 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga 7081 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc 7141 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat 7201 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg 7261 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga 7321 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc 7381 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg 7441 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc 7501 gaccgaaagg agcgcacgac cccatgcatc tcgagggccc ggtaccttta agaccaatga 7561 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 7621 tagctcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag 7681 accagatctg agcctgggag ctctctggct gcctagggaa cccactgctt aagcctcaat 7741 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 7801 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc 7861 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact 7921 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 7981 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 8041 atgtctggct ctagctatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 8101 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 8161 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcgtcgaga cgtacccaat 8221 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac 8281 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 8341 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 8401 ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 8461 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 8521 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 8581 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 8641 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 8701 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 8761 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 8821 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc c //