LOCUS       sequence-407772-        8062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Terminal deoxynucleotidyl transferase (TdT) 
            cysteine variant. Only one surface-exposed cysteine, at residue 302.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8062)
  AUTHORS   Palluk S, Arlow DH, de Rond T, Barthel S, Kang JS, Bector R, 
            Baghdassarian HM, Truong AN, Kim PW, Singh AK, Hillson NJ, Keasling 
            JD
  TITLE     De novo DNA synthesis using polymerase-nucleotide conjugates.
  JOURNAL   Nat Biotechnol. 2018 Aug;36(7):645-650. doi: 10.1038/nbt.4173. Epub 
            2018 Jun 18.
  PUBMED    29912208
REFERENCE   2  (bases 1 to 8062)
  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"; doi: "10.1038/nbt.4173"; 
            journalName: "Nat Biotechnol"; date: "2018-08"; volume: "36"; issue:
            "7"; pages: "645-650"
COMMENT     Sequence Label: pET19-MTdTc302
FEATURES             Location/Qualifiers
     source          1..8062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2013..2152
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2078..2097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2238..2260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2254..2445)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4800..4819
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4839..4916)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5122..5141
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5225..5244
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5225..5243
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5244..5268
                     /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             5283..5305
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5318..5347
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5363..5377
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             5384..6484
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     6458..6481
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6533..6544
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7789..7807)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7803..7850
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8025..8030,8031..8047,8048..8053))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgccaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggagag tgcctgatgc ggtattttct ccttacgcat
     2041 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2101 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2161 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2221 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2281 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2341 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2401 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2461 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2521 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2581 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2641 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2701 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     2761 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     2821 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     2881 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     2941 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     3001 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
     3061 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
     3121 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
     3181 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg
     3241 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
     3301 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
     3361 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
     3421 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3481 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3541 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3601 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     3661 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3721 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3781 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgcc agggtggttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3901 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3961 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4021 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4081 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4141 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4201 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4261 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4321 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4381 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4441 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4501 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4561 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4621 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4681 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4741 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4801 gcatactctg cgacatcgta taacgttact ggtttcacat taaccaccct gaattgactc
     4861 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4921 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4981 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5041 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5101 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5161 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     5221 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5281 attttgttta actttaagaa ggagatatac catgggccat catcatcatc atcatcatca
     5341 tcatcacagc agcggccata tcgacgacga cgacaagcat atgatgaaaa tcgaagaagg
     5401 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     5461 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     5521 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     5581 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     5641 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     5701 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     5761 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     5821 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     5881 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     5941 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     6001 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     6061 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     6121 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     6181 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     6241 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     6301 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     6361 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     6421 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     6481 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgaggg
     6541 aaggatttca catatgtcca tgggcggccg cgatatcgtc gacggatccg aattctcacc
     6601 gagtcccgtt ccgggtagcc agaacgtccc tgctccggcc gtgaagaaga tctcgcagta
     6661 cgcctgccaa cggcggacca ctcttaataa ttataaccaa ctgtttacag atgcgctgga
     6721 catcttagct gaaaacgatg agttgcgtga gaacgaggga agtgcccttg ctttcatgcg
     6781 tgcgtcatca gttctgaaga gtttaccttt ccctataacg agtatgaaag acacggaggg
     6841 cattccctcc ttaggggaca aagttaagtc tattatagaa gggataattg aggacggtga
     6901 gagttcagaa gcgaaggcgg tactgaatga cgagcgttac aaatcgttca agctgtttac
     6961 atcggtcttc ggtgtgggat tgaagactgc cgaaaagtgg tttagaatgg gctttagaac
     7021 cttgagcaag atacagagcg acaagtcgct gcgtttcacg caaatgcaaa aggcaggttt
     7081 cttgtactac gaggacctgg tctcctgtgt taatagacct gaggccgagg cggtctccat
     7141 gcttgtaaaa gaagcagttg ttacgttttt gccggatgct ttggtgacca tgaccggcgg
     7201 cttccgtaga gggaagatga ctgggcatga tgtggatttc ctgattacct ccccggaggc
     7261 cacggaggac gaagagcagc agttacttca taaagtcaca gatttctgga aacagcaagg
     7321 gcttttactt tatgccgaca tcttggagtc tacatttgaa aagtttaaac aaccatcgcg
     7381 caaagttgat gctcttgacc actttcagaa gtgcttcctg atattgaagt tggatcatgg
     7441 tcgggtgcac tcagagaagt ctggccagca ggagggcaaa ggctggaagg caatacgggt
     7501 tgatcttgtg atgtctccct atgaccgtag agcctttgca ttgcttgggt ggacgggttc
     7561 ccgccaattt gaaagagatt taagacgtta tgccacccac gaaagaaaaa tgatgctgga
     7621 taaccacgca ctgtatgacc gcacaaagcg ggtgtttctt gaggcggagt cagaagaaga
     7681 gatctttgcg cacttaggtt tagactacat cgagccgtgg gaacgtaacg cctaataagg
     7741 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     7801 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     7861 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc
     7921 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca
     7981 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat
     8041 gataagctgt caaacatgag aa
//
