LOCUS       sequence-434085-        6337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes codon optimized TfCut2 L32E-S113E-T237Q enzyme for bacterial
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  AUTHORS   Norton-Baker B, Denton MCR, Murphy NP, Fram B, Lim S, Erickson E, 
            Gauthier NP, Beckham GT
  TITLE     Enabling high-throughput enzyme discovery and engineering with a 
            low-cost, robot-assisted pipeline.
  JOURNAL   Sci Rep. 2024 Jun 24;14(1):14449. doi: 10.1038/s41598-024-64938-0.
  PUBMED    38914665
REFERENCE   2  (bases 1 to 6337)
  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: "Sci Rep."; 
            date: "2024-06-24"; volume: "14(1):14449. doi"; pages: " 
            10.1038/s41598-024-64938-0"
COMMENT     Sequence Label: pCDB179-TfCut2-L32E-S113E-T237Q
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(61..79)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      75..122
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      159..614
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(246..265)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     456..477
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(706..1521)
                     /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(706..1521)
                     /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     1429..1448
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1513..1532
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1643..2231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2132..2151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2385..2402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2417..2556
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2482..2501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2642..2664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2658..2849)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3658..4740)
                     /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     4702..4721
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4741..4818)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5024..5043
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5131..5150
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5131..5149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5154..5170
                     /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             5189..5211
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5225..5254
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 taatgatcga ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
       61 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      121 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      181 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      241 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      301 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      361 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      421 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      481 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      541 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
      601 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      661 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
      721 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
      781 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
      841 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
      901 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
      961 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1021 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1081 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1141 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1201 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1261 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1321 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1381 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1441 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     1501 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     1561 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1621 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1681 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1741 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     1801 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1861 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1921 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1981 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2041 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2101 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2161 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2221 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2281 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2341 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2401 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2461 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     2521 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     2581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     2701 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     2761 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     2821 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     2881 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     2941 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3001 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3061 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3121 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3181 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3241 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3301 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     3361 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     3421 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     3481 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     3541 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     3601 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     3661 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3721 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     3781 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     3841 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     3901 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     3961 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4021 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4081 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4141 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4201 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4261 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4321 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4381 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4441 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4501 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4561 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4621 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     4681 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     4741 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     4801 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     4861 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     4921 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     4981 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5041 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5101 ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg aaattgtgag
     5161 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
     5221 gggtcaccac caccaccacc accaccacca ccacggtagc ctgcaagatt cggaagtcaa
     5281 ccaagaagca aagccggaag tcaagccgga agtgaaaccg gaaacccata ttaacctgaa
     5341 agttagtgac ggcagctctg aaattttctt taagatcaaa aagaccacgc cgctgcgtcg
     5401 cctgatggaa gcgtttgcca aacgtcaggg caaggaaatg gatagcctgc gtttcctgta
     5461 tgacggtatt cgcatccagg cagatcaagc gccggaagac ctggacatgg aagacaacga
     5521 catcattgaa gcccaccgtg aacagattgg cggcgcgaac ccttacgaac gcggtccgaa
     5581 cccgaccgat gccctgctcg aagctcgctc gggcccgttc tctgtctccg aagaaaacgt
     5641 gagccgtgaa tcggcttccg gctttggcgg tggcacaatt tactatcctc gcgagaacaa
     5701 cacctacggt gctgttgcga tctctccggg ctatactggt acagaggctt ccatcgcctg
     5761 gctgggcgag cgcatcgctt ctcacggttt cgttgtcatt accatcgata ctattaccac
     5821 cctggaccag ccggactcgc gtgctgaaca gcttaatgca gcgcttaacc atatgatcaa
     5881 tcgtgcttcg gaaaccgttc gcagccgtat cgattcttct cgtctggcgg tgatgggtca
     5941 ttctatgggt ggcggtggtt cgctccgtct ggccagccag cgcccggatc tgaaagcggc
     6001 aatcccgctg actccgtggc atctgaacaa aaactggtct tcggttaccg tgccgaccct
     6061 gattatcggt gcagacctgg acacgattgc accggttgcg actcacgcaa aaccgttcta
     6121 caactccctg ccgtcttcta tttctaaggc ataccttgaa ctggacggtg caacccattt
     6181 cgctccgaac attccgaaca aaatcatcgg taaatacagc gtggcctggc tgaaacgttt
     6241 tgttgacaac gacacccgtt accagcagtt cctgtgcccg ggtccgcgtg acggtctttt
     6301 cggcgaggtg gaagaatatc gtagcacctg tccattc
//
