LOCUS Exported 7211 bp ds-DNA circular SYN 05-JUL-2018 DEFINITION . ACCESSION . VERSION . KEYWORDS pcDNA5FRTTO-Puro-eGFP-HEC1-d80 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7211) AUTHORS . TITLE Direct Submission JOURNAL Exported 5 Jul 2018 from SnapGene 2.3.2 http://www.snapgene.com COMMENT This file is created by Vector NTI http://www.invitrogen.com/ FEATURES Location/Qualifiers source 1..7211 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 235..590 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 591..958 /label="CMV promoter (with TetO's)" /note="CMV promoter" primer_bind 769..789 /note="CMV forward primer" protein_bind 820..838 /gene="tetO" /bound_moiety="tetracycline repressor TetR" /note="tet operator" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /bound_moiety="tetracycline repressor TetR" /note="tet operator" /note="bacterial operator O2 for the tetR and tetA genes" CDS 1080..1796 /codon_start=1 /product="enhanced GFP" /note="EGFP" /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1812..1814 /codon_start=1 /label="Hec1 9A" /note="Hec1 9A" /translation="M" CDS 1815..3503 /codon_start=1 /label="Hec1" /note="Hec1" /translation="IKDPRPLNDKAFIQQCIRQLCEFLTENGYAHNVSMKSLQAPSVKD FLKIFTFLYGFLCPSYELPDTKFEEEVPRIFKDLGYPFALSKSSMYTVGAPHTWPHIVA ALVWLIDCIKIHTAMKESSPLFDDGQPWGEETEDGIMHNKLFLDYTIKCYESFMSGADS FDEMNAELQSKLKDLFNVDAFKLESLEAKNRALNEQIARLEQEREKEPNRLESLRKLKA SLQGDVQKYQAYMSNLESHSAILDQKLNGLNEEIARVELECETIKQENTRLQNIIDNQK YSVADIERINHERNELQQTINKLTKDLEAEQQKLWNEELKYARGKEAIETQLAEYHKLA RKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRAQVYVPLKELLNETEEEINKALNK KMGLEDTLEQLNAMITESKRSVRTLKEEVQKLDDLYQQKIKEAEEEDEKCASELESLEK HKHLLESTVNQGLSEAMNELDAVQREYQLVVQTTTEERRKVGNNLQRLLEMVATHVGSV EKHLEEQIAKVDREYEECMSEDLSENIKEIRDKYEKKATLIKSSEE" polyA_signal 3555..3779 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" protein_bind 4062..4109 /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FRT" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 4153..4749 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /note="PuroR" /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" polyA_signal 4885..5005 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" primer_bind complement(5054..5070) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 5078..5094 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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 complement(5102..5132) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 5147..5168 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5456..6044) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6215..7075) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7076..7180) /gene="bla" /note="AmpR promoter" ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga 1021 attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg gtcgccacca 1081 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 1141 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 1201 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 1261 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 1321 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 1381 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 1441 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 1501 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 1561 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 1621 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 1681 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 1741 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg 1801 gactcagatc catgatcaag gacccgagac cacttaacga caaagcattc attcagcagt 1861 gtattcgaca actctgtgag tttcttacag aaaatggtta tgcacataat gtgtccatga 1921 aatctctaca agctccctct gttaaagact tcctgaagat cttcacattt ctttatggct 1981 tcctgtgccc ctcatacgaa cttcctgaca caaagtttga agaagaggtt ccaagaatct 2041 ttaaagacct tgggtatcct tttgcactat ccaaaagctc catgtacaca gtgggggctc 2101 ctcatacatg gcctcacatt gtggcagcct tagtttggct aatagactgc atcaagatac 2161 atactgccat gaaagaaagc tcacctttat ttgatgatgg gcagccttgg ggagaagaaa 2221 ctgaagatgg aattatgcat aataagttgt ttttggacta caccataaaa tgctatgaga 2281 gttttatgag tggtgccgac agctttgatg agatgaatgc agagctgcag tcaaaactga 2341 aggatttatt taatgtggat gcttttaagc tggaatcatt agaagcaaaa aacagagcat 2401 tgaatgaaca gattgcaaga ttggaacaag aaagagaaaa agaaccgaat cgtctagagt 2461 cgttgagaaa actgaaggct tccttacaag gagatgttca aaagtatcag gcatacatga 2521 gcaatttgga gtctcattca gccattcttg accagaaatt aaatggtctc aatgaggaaa 2581 ttgctagagt agaactagaa tgtgaaacaa taaaacagga gaacactcga ctacagaata 2641 tcattgacaa ccagaagtac tcagttgcag acattgagcg aataaatcat gaaagaaatg 2701 aattgcagca gactattaat aaattaacca aggacctgga agctgaacaa cagaagttgt 2761 ggaatgagga gttaaaatat gccagaggca aagaagcgat tgaaacacaa ttagcagagt 2821 atcacaaatt ggctagaaaa ttaaaactta ttcctaaagg tgctgagaat tccaaaggtt 2881 atgactttga aattaagttt aatcccgagg ctggtgccaa ctgccttgtc aaatacaggg 2941 ctcaagttta tgtacctctt aaggaactcc tgaatgaaac tgaagaagaa attaataaag 3001 ccctaaataa aaaaatgggt ttggaggata ctttagaaca attgaatgca atgataacag 3061 aaagcaagag aagtgtgaga actctgaaag aagaagttca aaaattggat gatctttacc 3121 aacaaaaaat taaggaagca gaggaagagg atgaaaaatg tgccagtgag cttgagtcct 3181 tggagaaaca caagcacctg ctagaaagta ctgttaacca ggggctcagt gaagctatga 3241 atgaattaga tgctgttcag cgggaatacc aactagttgt gcaaaccacg actgaagaaa 3301 gacgaaaagt gggaaataac ttgcaacgtc tgttagagat ggttgctaca catgttgggt 3361 ctgtagagaa acatcttgag gagcagattg ctaaagttga tagagaatat gaagaatgca 3421 tgtcagaaga tctctcggaa aatattaaag agattagaga taagtatgag aagaaagcta 3481 ctctaattaa gtcttctgaa gaatgactcg aggacggtac cgcgggcccg tttaaacccg 3541 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 3601 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 3661 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 3721 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 3781 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 3841 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 3901 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 3961 ccgtcaagct ctaaatcggg ggtcccttta gggttccgat ttagtgcttt acggcacctc 4021 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt 4081 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg 4141 tgctggcgcg ccaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 4201 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 4261 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 4321 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc 4381 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 4441 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg 4501 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag 4561 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc 4621 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc 4681 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 4741 ggtgcctgac ctaggcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg 4801 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 4861 atgctggagt tcttcgccca ccccaacttg tttattgcag cttaaatggt tacaaataaa 4921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 4981 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct 5041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 5101 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 5161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 5281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 5761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 5821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 5881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 5941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 6781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 6841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 6901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 6961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7201 cacctgacgt c //