LOCUS       pRK5R-99xGly-EGFP-3xHA  5848 bp ds-DNA     circular     15-MAR-2024
DEFINITION  .
FEATURES             Location/Qualifiers
     rep_origin      complement(1..589)
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
                     /direction="RIGHT"
     primer_bind     complement(81..100)
                     /label="pBR322ori-F"
                     /note="pBR322 origin, forward primer"
     CDS             complement(760..1620)
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     1383..1402
                     /label="Amp-R"
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /label="AmpR promoter"
                     /gene="bla"
     primer_bind     1793..1811
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward primer"
     primer_bind     complement(1849..1871)
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     primer_bind     1971..1990
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable marker"
     rep_origin      complement(2007..2462)
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
                     /direction="RIGHT"
     primer_bind     complement(2144..2165)
                     /label="F1ori-F"
                     /note="F1 origin, forward primer"
     primer_bind     2356..2375
                     /label="F1ori-R"
                     /note="F1 origin, reverse primer"
     primer_bind     2660..2682
                     /label="M13/pUC Forward"
                     /note="In lacZ gene"
     primer_bind     2674..2691
                     /label="M13 Forward"
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward"
     primer_bind     2675..2691
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar variants"
     enhancer        2711..3090
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3091..3294
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early promoter"
     primer_bind     3245..3265
                     /label="CMV-F"
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3291..3315
                     /label="LNCX"
                     /note="Human CMV promoter, forward primer"
     primer_bind     3525..3542
                     /label="SP6"
                     /note="SP6 promoter, forward primer"
     promoter        3525..3543
                     /label="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3556..3575
                     /label="pMT2-F"
                     /note="Synthetic intron, forward primer"
     primer          complement(3621..3651)
                     /label="oJY120"
                     /note="sequence: gcctcCATGGTGGCGAATTCGAATTAATTAAACCG"
     misc_feature    3642..3647
                     /label="Kozak"
     CDS             3648..4826
                     /label="Translation 3648-4826"
                     /translation="MGLPLPGGVRQRGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGWASTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGYPYDVPDYAGTGSYPYDVPDYAGSAGYPYDVPDYA*"
     primer          complement(3659..3673)
                     /label="RC443"
                     /note="sequence: acgccccctggcagc"
     misc_feature    3684..3980
                     /label="99xGly"
     misc_feature    3993..4706
                     /label="EGFP"
     primer_bind     complement(4035..4056)
                     /label="EGFP-N"
     primer_bind     complement(4296..4315)
                     /label="EXFP-R"
     primer_bind     4643..4664
                     /label="EGFP-C"
     primer          4713..4732
                     /label="oJY119"
                     /note="sequence: atcgatgcggccgctAGCGGCTATCCCTATGACGT"
     misc_feature    4719..4823
                     /label="3xHA"
     polyA_signal    4857..4991
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4894..4913)
                     /label="SV40pA-R"
                     /note="SV40 polyA, reverse primer"
     primer_bind     4948..4967
                     /label="EBV-rev"
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5060..5417
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(5083..5103)
                     /label="pBABE 3'"
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5268..5403
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     primer_bind     5330..5349
                     /label="SV40pro-F"
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5447..5463)
                     /label="M13 Reverse"
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5447..5463)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar variants"
     primer_bind     complement(5460..5482)
                     /label="M13/pUC Reverse"
                     /note="In lacZ gene"
     protein_bind    complement(5471..5487)
                     /label="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)."
                     /bound_moiety="lac repressor encoded by lacI"
     promoter        complement(5495..5525)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5540..5561)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence of cAMP."
                     /bound_moiety="E. coli catabolite activator protein"
     primer_bind     complement(5678..5695)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     1801 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     1861 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     1921 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     1981 gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc
     2041 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2101 ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2161 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2221 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     2281 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2341 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2401 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2461 gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     2521 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     2581 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     2641 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag taattaattc
     2701 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2761 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2821 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2881 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2941 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     3001 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     3061 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     3121 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     3181 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     3241 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
     3301 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
     3361 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
     3421 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
     3481 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     3541 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
     3601 tcggttctag ctagcgctac cggtttaatt aattcgAATT CgccaccATG GGGTTgccgc
     3661 tgccaggggg cgtgcggcag cgcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3721 gcggaggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3781 gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3841 gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3901 gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg
     3961 gcggcggcgg cggcggcggc tgggccTCGA CGgtgagcaa gggcgaggag ctgttcaccg
     4021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     4081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     4141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     4201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     4261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4681 ctctcggcat ggacgagctg tacaagggag ggagcggcta tccctatgac gtgcctgatt
     4741 acgccggcac aggatcatac ccctatgatg tgcctgacta cgctggcagc gccggatacc
     4801 cttatgatgt gcctgattat gctTAGtgag gatccccagc ttggccgcca tggcccaact
     4861 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4921 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4981 atgtctggat cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag
     5041 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     5101 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5161 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5221 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     5281 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     5341 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     5401 gcctaggctt ttgcaaaaag ctgttaacag cttggctcgt aatcatgtca tagctgtttc
     5461 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5521 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5581 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5641 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5701 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5761 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5821 accgtaaaaa ggccgcgttg ctggcgtt
//