LOCUS       sequence-309541-        5664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for the de novo designed protein 
            Expression_vector_NT_1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5664)
  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 5664)
  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_1
FEATURES             Location/Qualifiers
     source          1..5664
                     /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             529..546
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(599..617)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      613..660
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      697..1152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(784..803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     994..1015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1244..2059)
                     /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(1244..2059)
                     /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     1967..1986
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2051..2070
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2181..2769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2670..2689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2923..2940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2955..3094
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3020..3039)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3180..3202
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3196..3387)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4196..5278)
                     /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     5240..5259
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5279..5356)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5562..5581
                     /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 tcgcgcgagg agatccgtaa agtagtcgaa
      181 acctttctgc gtgctgcgaa tagccaggac aaaaaaaaac tcgaagaggc ggcgaaaaat
      241 attctgtcac ctgatgttcg tctggaagtc ggcaactata cctggaccag cattgaacaa
      301 atgcttaagt tttatcaact gtctgagatt gatcgcgtcg aaattcgcaa agtgcaggtc
      361 gatggcaacc acgtgcgtgt ggaaattgaa gtagaacgta atggcaaaaa gtggacctgg
      421 gaagttgagg tggaagtacg taatggtttg attaaacgca ttcgtaatca ggtcgatccg
      481 gaatataaaa aagatgtgca aaatatctgg aataatacct aactcgagca ccaccaccac
      541 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      601 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      661 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      721 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      781 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      841 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      901 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      961 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1021 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1081 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1141 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1201 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     1261 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1321 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1381 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1441 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1501 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1561 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1621 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1681 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1741 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     1801 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     1861 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     1921 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     1981 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2041 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2101 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2761 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2821 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2881 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2941 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3001 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3061 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3121 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3181 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3241 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3301 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3361 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3421 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3481 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3541 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3601 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3661 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3721 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3781 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3841 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     3901 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     3961 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4021 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4081 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4141 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4201 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4261 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4321 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4381 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4441 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4501 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4561 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4621 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4681 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4741 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4801 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4861 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     4921 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     4981 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5041 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5101 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5161 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5221 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5281 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5341 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5401 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5461 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5521 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     5581 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     5641 atcgagatct cgatcccgcg aaat
//
