LOCUS       pET28a-ImpBeta1-        8557 bp DNA     circular SYN 09-JAN-2025
DEFINITION  Encodes Importin Beta1 (human) with a C-terminal mEosEM tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8557)
  AUTHORS   Siegfried Musser
  TITLE     Direct Submission
  JOURNAL   Exported Jan 9, 2025 from SnapGene 7.2.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8557
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     protein_bind    5003..5027
                     /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)."
                     /note="color: #31849b"
     RBS             5042..5064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     primer_bind     5064..5100
                     /label=Primer 1 (2)
                     /note="color: black; sequence: 
                     ATATACCATGGAGCTGATCACCATTCTCGAGAAGACC; added: 2023-11-28"
     gene            5071..7698
                     /label=Importin Beta1
                     /note="color: #ff0000; direction: RIGHT"
     primer_bind     5071..5092
                     /label=Primer 1
                     /note="color: black; sequence: 
                     CGCTACCGGTATGGAGCTGATCACCATTCTCG; added: 2023-11-28"
     primer_bind     complement(7672..7704)
                     /label=Primer 2
                     /note="color: black; sequence: 
                     CCCAATTGAGCTTGGTTCTTCAGTTTCCTCAGTTC; added: 2023-11-28"
     primer_bind     7694..7745
                     /label=Primer 1 (1)
                     /note="color: black; sequence: 
                     AAGCTCAATTGGGAGGAAGTGCGATTAAGCCAGACATGAAGATCAAACTCCG; 
                     added: 2023-11-28"
     primer_bind     7698..7724
                     /label=F-Beta1-MfeI-mEosEM
                     /note="color: black; sequence: 
                     ATATCAATTGatggtgagtgcgattaagcc; added: 2023-11-28"
     CDS             7705..8385
                     /codon_start=1
                     /product="fixation-resistant truly monomeric green-to-red 
                     photoswitchable fluorescent protein (Paez-Segala et al., 
                     2015)"
                     /label=mEos4b
                     /note="derived from mEos2"
                     /note="color: #00ff00"
                     /translation="MVSAIKPDMRIKLRMEGNVNGHHFVIDGEGTGKPYEGKQTMDLEV
                     KEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSMTFEDGGICNA
                     RNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALL
                     LEGGAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLP
                     DNARR"
     primer_bind     complement(8367..8397)
                     /label=Primer 2 (2)
                     /note="color: black; sequence: 
                     GAGTGCGGCCGCTCGTCTGGCATTGTCAGGC; added: 2023-11-28"
     primer_bind     complement(8367..8394)
                     /label=R-Beta1-NotI-mEosEM
                     /note="color: black; sequence: 
                     ATATGCGGCCGCtcgtctggcattgtcaggc; added: 2023-11-28"
     CDS             8401..8418
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      8485..8532
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atggagctga tcaccattct cgagaagacc
     5101 gtgtctcccg atcggctgga gctggaagcg gcgcagaagt tcctggagcg tgcggccgtg
     5161 gagaacctgc ccactttcct tgtggaactg tccagagtgc tggcaaatcc aggaaacagt
     5221 caggttgcca gagttgcagc tggtctacaa atcaagaact ctttgacatc taaagatcca
     5281 gatatcaagg cacaatatca gcagaggtgg cttgctattg atgctaatgc tcgacgagaa
     5341 gtcaagaact atgttttgca gacattgggt acagaaactt accggcctag ttctgcctca
     5401 cagtgtgtgg ctggtattgc ttgtgcagag atcccagtaa accagtggcc agaactcatt
     5461 cctcagctgg tggccaatgt cacaaacccc aacagcacag agcacatgaa ggagtcgaca
     5521 ttggaagcca tcggttatat ttgccaagat atagacccag agcagctaca agataaatcc
     5581 aatgagattc tgactgccat aatccagggg atgaggaaag aagagcctag taataatgtg
     5641 aagctagctg ctacgaatgc actcctgaac tcattggagt tcaccaaagc aaactttgat
     5701 aaagagtctg aaaggcactt tattatgcag gtggtctgtg aagccacaca gtgtccagat
     5761 acgagggtac gagtggctgc tttacagaat ctggtgaaga taatgtcctt atattatcag
     5821 tacatggaga catatatggg tcctgctctt tttgcaatca caatcgaagc aatgaaaagt
     5881 gacattgatg aggtggcttt acaagggata gaattctggt ccaatgtctg tgatgaggaa
     5941 atggatttgg ccattgaagc ttcagaggca gcagaacaag gacggccccc tgagcacacc
     6001 agcaagtttt atgcgaaggg agcactacag tatctggttc caatcctcac acagacacta
     6061 actaaacagg acgaaaatga tgatgacgat gactggaacc cctgcaaagc agcaggggtg
     6121 tgcctcatgc ttctggccac ctgctgtgaa gatgacattg tcccacatgt cctccccttc
     6181 attaaagaac acatcaagaa cccagattgg cggtaccggg atgcagcagt gatggctttt
     6241 ggttgtatct tggaaggacc agagcccagt cagctcaaac cactagttat acaggctatg
     6301 cccaccctaa tagaattaat gaaagacccc agtgtagttg ttcgagatac agctgcatgg
     6361 actgtaggca gaatttgtga gctgcttcct gaagctgcca tcaatgatgt ctacttggct
     6421 cccctgctac agtgtctgat tgagggtctc agtgctgaac ccagagtggc ttcaaatgtg
     6481 tgctgggctt tctccagtct ggctgaagct gcttatgaag ctgcagacgt tgctgatgat
     6541 caggaagaac cagctactta ctgcttatct tcttcatttg aactcatagt tcagaagctc
     6601 ctagagacta cagacagacc tgatggacac cagaacaacc tgaggagttc tgcatatgaa
     6661 tctctgatgg aaattgtgaa aaacagtgcc aaggattgtt atcctgctgt ccagaaaacg
     6721 actttggtca tcatggaacg actgcaacag gttcttcaga tggagtcaca tatccagagc
     6781 acatccgata gaatccagtt caatgacctt cagtctttac tctgtgcaac tcttcagaat
     6841 gttcttcgga aagtgcaaca tcaagatgct ttgcagatct ctgatgtggt tatggcctcc
     6901 ctgttaagga tgttccaaag cacagctggg tctgggggag tacaagagga tgccctgatg
     6961 gcagttagca cactggtgga agtgttgggt ggtgaattcc tcaagtacat ggaggccttt
     7021 aaacccttcc tgggcattgg attaaaaaat tatgctgaat accaggtttg tttggcagct
     7081 gtgggcttag tgggagactt gtgccgtgcc ctgcaatcca acatcatacc tttctgtgac
     7141 gaggtgatgc agctgcttct ggaaaatttg gggaatgaga acgtccacag gtctgtgaag
     7201 ccgcagattc tgtcagtgtt tggtgatatt gcccttgcta ttggaggaga gtttaaaaaa
     7261 tacttagagg ttgtattgaa tactcttcag caggcctccc aagcccaggt ggacaagtca
     7321 gactatgaca tggtggatta tctgaatgag ctaagggaaa gctgcttgga agcctatact
     7381 ggaatcgtcc agggattaaa gggggatcag gagaacgtac acccggatgt gatgctggta
     7441 caacccagag tagaatttat tctgtctttc attgaccaca ttgctggaga tgaggatcac
     7501 acagatggag tagtagcttg tgctgctgga ctaatagggg acttatgtac agcatttggg
     7561 aaggatgtac tgaaattagt agaagctagg ccaatgatcc atgaattgtt aactgaaggg
     7621 cggagatcga agactaacaa agcaaaaacc cttgctacat gggcaacaaa agaactgagg
     7681 aaactgaaga accaagctca attgatggtg agtgcgatta agccagacat gaggatcaaa
     7741 ctccgtatgg aaggcaacgt aaacgggcac cactttgtga tcgacggaga aggtacaggc
     7801 aagccttatg agggaaaaca gaccatggat cttgaagtca aagagggcgg acctctgcct
     7861 tttgcctttg atatcctgac cactgcattc cattacggca acagggtatt cgtgaaatat
     7921 ccagacaaca tacaagacta ttttaagcag tcgtttccta aggggtattc gtgggaacga
     7981 agcatgactt tcgaagacgg gggcatttgc aatgccagaa acgacataac aatggaaggg
     8041 gacactttct ataataaagt tcgattttat ggtaccaact ttcccgccaa tggtccagtt
     8101 atgcagaaga agacgctgaa atgggagccc tccactgaga aaatgtatgt gcgtgatgga
     8161 gtgctgacgg gtgatattga gatggctttg ttgcttgaag gaggtgccca ttaccgatgt
     8221 gacttcagaa ctacttacaa agctaaggag aagggtgtca agttaccagg cgcccacttt
     8281 gtggaccacg ccattgagat tttaagccat gacaaagatt acaacaaggt taagctgtat
     8341 gagcatgctg ttgctcattc tggattgcct gacaatgcca gacgagcggc cgcactcgag
     8401 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     8461 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     8521 aggggttttt tgctgaaagg aggaactata tccggat
//
