LOCUS       sequence-323634-        6150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mature human Transforming Growth Factor alpha fused to SPM
            in bacterial cells for calcium-induced covalent coupling to 
            Epidermal Growth Factor Receptor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  AUTHORS   Scheu AHA, Lim SYT, Metzner FJ, Mohammed S, Howarth M
  TITLE     NeissLock provides an inducible protein anhydride for covalent 
            targeting of endogenous proteins.
  JOURNAL   Nat Commun. 2021 Jan 29;12(1):717. doi: 10.1038/s41467-021-20963-5.
  PUBMED    33514717
REFERENCE   2  (bases 1 to 6150)
  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: "Nat 
            Commun."; date: "2021-01-29"; volume: "12(1):717. doi"; pages: " 
            10.1038/s41467-021-20963-5"
COMMENT     Sequence Label: pET28a-TGF-alpha-GSY-SPM
FEATURES             Location/Qualifiers
     source          1..6150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             991..1008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1085..1103)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1099..1146
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1183..1611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1270..1289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1480..1501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1730..2545)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1730..2545)
                     /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"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2453..2472
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2537..2556
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2667..3255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3156..3175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3409..3426
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3441..3580
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3506..3525)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3666..3688
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3682..3873)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4682..5764)
                     /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     5726..5745
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5765..5842)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6048..6067
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gtcgtaagcc attttaatga ctgtcctgac
      121 tcacacaccc agttctgttt tcacggcact tgtcgcttct tagtgcaaga agacaagcca
      181 gcgtgtgtct gccatagtgg gtatgttggg gctcgttgcg aacatgctga ccttcttgcc
      241 ggctcgtatg atccactggc attagatctt gatggcgacg gtatcgaaac ggtggcaacg
      301 aaggggtttg ccggctcctt attcgatcac acaaataacg gaattcgcac agctactgga
      361 tgggtaagtg ccgacgacgg cctgttggtg cgcgacctga atggcaacgg gattatcgac
      421 aatggcgcgg aactttttgg agataacacg aaattggcag acgggagttt tgctaagcac
      481 gggtatgcgg cgttggcgga gttggatagt aacggagaca atatcatcaa tgcggcagat
      541 gcggcattcc agactttgcg cgtgtggcaa gatctgaatc aggacggtat ttcccaagct
      601 aatgagcttc gtacattgga ggagcttggt atccagtcgt tagacctggc atacaaagat
      661 gtcaacaaga atttaggtaa cggtaatact ttagctcagc aaggtagcta caccaaaacc
      721 gacggaacca ctgcaaaaat gggcgacttg ttgctggcag cagataatct gcactcgcgt
      781 ttcaaggaca aagtagaact tactgcggaa caggcaaagg ccgcaaatct ggccggtatt
      841 ggtcgtttac gcgacttacg cgaggccgca gctctttctg gagatctggc taatatgtta
      901 aaagcctatt cagccgcgga gaccaaagaa gcccaattgg ctctgttgga taatttgatt
      961 cacaaatggg ccgagacgga cggcagcagc catcatcacc atcaccacag cagcggcgag
     1021 ccagaggctt aataagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1081 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1141 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgscc tgtagcggcg
     1201 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1261 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1321 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1381 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1441 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1501 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1561 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1621 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1681 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1741 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1801 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1861 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1921 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1981 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2041 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2101 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2161 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2221 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2281 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2341 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2401 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     2461 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2521 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2581 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2641 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2701 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2761 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2821 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2881 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2941 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3001 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3061 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3121 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3181 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3241 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3301 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3361 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3421 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3481 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3541 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3601 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3661 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3721 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3781 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3841 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3901 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3961 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4021 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4081 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4141 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4201 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4261 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4321 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4381 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4441 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4501 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4561 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4621 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4681 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4741 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4801 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4861 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4921 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4981 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5041 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5101 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5161 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5221 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5281 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5341 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5401 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5461 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5521 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5581 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5641 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5701 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5761 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5821 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5881 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5941 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6001 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6061 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6121 tagaggatcg agatctcgat cccgcgaaat
//
