LOCUS       sequence-407817-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Terminal deoxynucleotidyl transferase (TdT) 
            cysteine variant. Only one surface-exposed cysteine, at residue 253.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  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 6851)
  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-TdTc253
FEATURES             Location/Qualifiers
     source          1..6851
                     /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"
     CDS             complement(3757..4839)
                     /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     4801..4820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4840..4917)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5123..5142
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5226..5245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5226..5244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5245..5269
                     /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             5284..5306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5319..5348
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5364..5378
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(6578..6596)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6592..6639
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6814..6819,6820..6836,6837..6842))
                     /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 ttttaattca 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 ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3901 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3961 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4021 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4081 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4141 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4201 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4261 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4321 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4381 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4441 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4501 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4561 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4621 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4681 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4741 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4801 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttaaccaccc tgaattgact
     4861 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4921 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4981 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5041 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5101 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5161 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5221 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     5281 aattttgttt aactttaaga aggagatata ccatgggcca tcatcatcat catcatcatc
     5341 atcatcacag cagcggccat atcgacgacg acgacaagca tatgtcaccg agtcccgttc
     5401 cgggtagcca gaacgtccct gctccggccg tgaagaagat ctcgcagtac gcctgccaac
     5461 ggcggaccac tcttaataat tataaccaac tgtttacaga tgcgctggac atcttagctg
     5521 aaaacgatga gttgcgtgag aacgagggaa gtgcccttgc tttcatgcgt gcgtcatcag
     5581 ttctgaagag tttacctttc cctataacga gtatgaaaga cacggagggc attccctcct
     5641 taggggacaa agttaagtct attatagaag ggataattga ggacggtgag agttcagaag
     5701 cgaaggcggt actgaatgac gagcgttaca aatcgttcaa gctgttttgt tcggtcttcg
     5761 gtgtgggatt gaagactgcc gaaaagtggt ttagaatggg ctttagaacc ttgagcaaga
     5821 tacagagcga caagtcgctg cgtttcacgc aaatgcaaaa ggcaggtttc ttgtactacg
     5881 aggacctggt ctccgctgtt aatagacctg aggccgaggc ggtctccatg cttgtaaaag
     5941 aagcagttgt tacgtttttg ccggatgctt tggtgaccat gaccggcggc ttccgtagag
     6001 ggaagatgac tgggcatgat gtggatttcc tgattacctc cccggaggcc acggaggacg
     6061 aagagcagca gttacttcat aaagtcacag atttctggaa acagcaaggg cttttacttt
     6121 atgccgacat cttggagtct acatttgaaa agtttaaaca accatcgcgc aaagttgatg
     6181 ctcttgacca ctttcagaag tgcttcctga tattgaagtt ggatcatggt cgggtgcact
     6241 cagagaagtc tggccagcag gagggcaaag gctggaaggc aatacgggtt gatcttgtga
     6301 tgtctcccta tgaccgtaga gcctttgcat tgcttgggtg gacgggttcc cgccaatttg
     6361 aaagagattt aagacgttat gccacccacg aaagaaaaat gatgctggat aaccacgcac
     6421 tgtatgaccg cacaaagcgg gtgtttcttg aggcggagtc agaagaagag atctttgcgc
     6481 acttaggttt agactacatc gagccgtggg aacgtaacgc ctaataagga tccggctgct
     6541 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     6601 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     6661 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     6721 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     6781 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     6841 aaacatgaga a
//
