LOCUS       sequence-418258-        6967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression of GFP labelled NEMO (SMC Internal No. 
            1977)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6967)
  TITLE     Sascha Martens Lab: ASAP Shared Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6967)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGEX-4T1-GST-Thrombin-TEV-EGFP-hNEMO
FEATURES             Location/Qualifiers
     source          1..6967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(82..87,88..103,104..110)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    118..134
                     /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)."
     CDS             157..810
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     768..790
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             817..834
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             835..855
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(862..864,865..867,868..1578)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(907..928)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1168..1187)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1515..1536
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2916..2938)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3083..3101)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3169..3273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3274..3342,3343..4134)
                     /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     complement(3492..3511)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4305..4893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4794..4813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5047..5064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5137..5214
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5215..6297
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5234..5253)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6346..6351,6352..6369,6370..6376)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6384..6400
                     /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     6408..6424
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6408..6424
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6420..6947
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6440..6457)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6440..6456)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6449..6471)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
       61 caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg
      121 tgagcggata acaatttcac acaggaaaca gtattcatgt cccctatact aggttattgg
      181 aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat
      241 gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg
      301 ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct
      361 atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag
      421 cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga
      481 attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa
      541 atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta
      601 acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg
      661 tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa
      721 attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc
      781 acgtttggtg gtggcgacca tcctccaaaa tcggatctgg ttccgcgtgg atccgaaaac
      841 ctgtattttc agggatcagg catggtgagc aagggcgagg agctgttcac cggggtggtg
      901 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      961 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1021 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     1081 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1141 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1201 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1261 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1321 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1381 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1441 gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac
     1501 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1561 atggacgagc tgtacaaggg cggtggaggc ttcaataggc acctctggaa gagccaactg
     1621 tgtgagatgg tgcagcccag tggtggcccg gcagcagatc aggacgtact gggcgaagag
     1681 tctcctctgg ggaagccagc catgctgcac ctgccttcag aacagggcgc tcctgagacc
     1741 ctccagcgct gcctggagga gaatcaagag ctccgagatg ccatccggca gagcaaccag
     1801 attctgcggg agcgctgcga ggagcttctg catttccaag ccagccagag ggaggagaag
     1861 gagttcctca tgtgcaagtt ccaggaggcc aggaaactgg tggagagact cggcctggag
     1921 aagctcgatc tgaagaggca gaaggagcag gctctgcggg aggtggagca cctgaagaga
     1981 tgccagcagc agatggctga ggacaaggcc tctgtgaaag cccaggtgac gtccttgctc
     2041 ggggagctgc aggagagcca gagtcgcttg gaggctgcca ctaaggaatg ccaggctctg
     2101 gagggtcggg cccgggcggc cagcgagcag gcgcggcagc tggagagtga gcgcgaggcg
     2161 ctgcagcagc agcacagcgt gcaggtggac cagctgcgca tgcagggcca gagcgtggag
     2221 gccgcgctcc gcatggagcg ccaggccgcc tcggaggaga agaggaagct ggcccagttg
     2281 caggtggcct atcaccagct cttccaagaa tacgacaacc acatcaagag cagcgtggtg
     2341 ggcagtgagc ggaagcgagg aatgcagctg gaagatctca aacagcagct ccagcaggcc
     2401 gaggaggccc tggtggccaa acaggaggtg atcgataagc tgaaggagga ggccgagcag
     2461 cacaagattg tgatggagac cgttccggtg ctgaaggccc aggcggatat ctacaaggcg
     2521 gacttccagg ctgagaggca ggcccgggag aagctggccg agaagaagga gctcctgcag
     2581 gagcagctgg agcagctgca gagggagtac agcaaactga aggccagctg tcaggagtcg
     2641 gccaggatcg aggacatgag gaagcggcat gtcgaggtct cccaggcccc cttgcccccc
     2701 gcccctgcct acctctcctc tcccctggcc ctgcccagcc agaggaggag cccccccgag
     2761 gagccacctg acttctgctg tcccaagtgc cagtatcagg cccctgatat ggacaccctg
     2821 cagatacatg tcatggagtg cattgagtag ctcgagcggc cgcatcgtga ctgactgacg
     2881 atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     2941 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3001 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     3061 tataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3121 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3181 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3241 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3301 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3361 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3421 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3481 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     3541 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3601 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3661 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3721 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3781 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     3841 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3901 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3961 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4021 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4081 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4141 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4201 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4261 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4321 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4381 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4441 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4501 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4561 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4621 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4681 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4741 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4801 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4861 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4921 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4981 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5041 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     5101 acgcatctgt gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc
     5161 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     5221 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     5281 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     5341 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     5401 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     5461 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     5521 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     5581 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     5641 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     5701 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     5761 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     5821 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     5881 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     5941 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     6001 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     6061 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     6121 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     6181 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     6241 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     6301 caacgcaatt aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct
     6361 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     6421 tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6481 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6541 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct
     6601 ttgcctggtt tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg
     6661 aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct
     6721 acaccaacgt gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga
     6781 cgggttgtta ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc
     6841 gaattatttt tgatggcgtt ggaattagct tatcgactgc acggtgcacc aatgcttctg
     6901 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
     6961 tcgtgtc
//
