LOCUS       sequence-309544-        5676 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_NT_9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5676)
  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 5676)
  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_9
FEATURES             Location/Qualifiers
     source          1..5676
                     /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             541..558
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(611..629)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      625..672
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      709..1164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(796..815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1006..1027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1256..2071)
                     /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(1256..2071)
                     /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     1979..1998
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2063..2082
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2193..2781
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2682..2701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2935..2952
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2967..3106
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3032..3051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3192..3214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3208..3399)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4208..5290)
                     /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     5252..5271
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5291..5368)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5574..5593
                     /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 agccgcgaag agatccgcaa agttgtcgag
      181 gagtatatcc gtctgctgta tacagaccct gatcagttca agaaagcggc ccgcgataaa
      241 ttgctgagtc cggatgtgcg tattgaaatc ggtaattata cgtttgattc ccgcaacctc
      301 gatcgctttc tggacgcaat gcaggaatgg gcgagccgtt acgatcgtgt ggaaattcgc
      361 aaggttcagg ttgacgggaa ccatgtgcgt gttgagattg agttggaatc gaacggtaaa
      421 aaatggacgt ttgaaatcga ggttgaggtt cgcaatggca aaattaagcg catccgtcaa
      481 caggtggatc cggaatataa aaaagttgtg cagaatctgt ggaataacac gtgactcgag
      541 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      601 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      661 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      721 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      781 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      841 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      901 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      961 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1021 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1081 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1141 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1201 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1261 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1321 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1381 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1441 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1501 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1561 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1621 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1681 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1741 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     1801 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1861 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1921 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1981 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2041 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2101 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2161 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2221 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2281 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2341 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2401 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2461 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2521 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2581 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2641 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2701 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2761 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2821 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2881 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2941 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3001 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3061 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3121 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3181 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3241 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3301 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3361 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3421 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3481 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3541 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3601 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3661 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     3721 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     3781 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     3841 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     3901 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     3961 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4021 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4081 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4141 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4201 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4261 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4321 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4381 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4441 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4501 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4561 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     4621 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     4681 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     4741 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     4801 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     4861 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     4921 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     4981 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5041 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5101 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5161 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5221 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     5281 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5341 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5401 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     5461 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     5521 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     5581 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     5641 ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
