LOCUS       sequence-309546-        5706 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_NT_10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5706)
  AUTHORS   Pan X, Thompson MC, Zhang Y, Liu L, Fraser JS, Kelly MJS, Kortemme T
  TITLE     Expanding the space of protein geometries by computational design of
            de novo fold families.
  JOURNAL   Science. 2020 Aug 28;369(6507):1132-1136. doi: 
            10.1126/science.abc0881.
  PUBMED    32855341
REFERENCE   2  (bases 1 to 5706)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2020-08-28- 28"; volume: "369"; 
            issue: "6507"; pages: "1132-1136"
COMMENT     Sequence Label: Expression_vector_NT_10
FEATURES             Location/Qualifiers
     source          1..5706
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             571..588
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(641..659)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      655..702
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      739..1194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(826..845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1036..1057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1286..2101)
                     /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(1286..2101)
                     /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     2009..2028
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2093..2112
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2223..2811
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2712..2731
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2965..2982
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2997..3136
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3062..3081)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3222..3244
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3238..3429)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4238..5320)
                     /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     5282..5301
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5321..5398)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5604..5623
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg tcccgggaag agattcggaa ggtcgtggag
      181 acttggatcc gcctttttta tagctcggat ccgaacgact gggaaacgtt ccagaaagcg
      241 aagaaagatc tgctctcacc agatgtgcgc gtagaaatcg gaaattacac gcttaatagt
      301 gaacaagtcg atcgttggtg ggaagcctgg gtgaaaatca tccagaaaga attggaagag
      361 aaaaacgaac cgctccgcac ggaaattcgc aaggttcaag tggacggcaa tcacgtacgc
      421 gtggaaatcg agcaggaaaa aaacggcaag aagtggacct ttgaagttga agtggaagtt
      481 cgtaatggaa aaatcaagcg tattcgtcaa caggtagacc cagagtacaa aaaggaggtc
      541 caggcggctt ggaacaacac ctaactcgag caccaccacc accaccactg agatccggct
      601 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      661 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      721 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      781 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1081 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1141 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1201 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1261 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1321 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1381 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1441 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1501 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1561 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1621 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     1681 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1741 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1801 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1861 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1921 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1981 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2041 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2101 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     2161 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2221 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2281 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2341 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2401 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2461 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2521 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2581 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2641 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2701 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2761 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2821 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2881 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2941 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3001 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3061 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3121 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3181 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3241 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3301 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3361 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     3421 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     3481 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     3541 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     3601 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3661 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3721 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3781 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3841 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3901 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3961 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     4021 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     4081 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     4141 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     4201 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4261 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4321 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     4381 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     4441 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     4501 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     4561 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4621 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4681 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4741 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4801 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4861 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4921 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4981 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     5041 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     5101 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     5161 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     5221 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5281 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     5341 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     5401 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     5461 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     5521 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     5581 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5641 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5701 cgaaat
//
