LOCUS       sequence-203224-        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  TITLE     Structural Genomics Consortium Clones
REFERENCE   2  (bases 1 to 6116)
  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"
COMMENT     Sequence Label: SIRT5
FEATURES             Location/Qualifiers
     source          1..6116
                     /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             981..998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1051..1069)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1065..1112
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1149..1604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1236..1255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1446..1467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1696..2511)
                     /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(1696..2511)
                     /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     2419..2438
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2503..2522
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2633..3221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3122..3141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3375..3392
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3407..3546
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3472..3491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3632..3654
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3648..3839)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4648..5730)
                     /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     5692..5711
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5731..5808)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6014..6033
                     /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 ttccgcgtgg tagtgctcgg ccaagttcaa gtatggcaga ttttcgaaag
      181 ttttttgcaa aagcaaagca catagtcatc atctcaggag ctggtgttag tgcagaaagt
      241 ggtgttccga ccttcagagg agctggaggt tattggagaa aatggcaagc ccaggacctg
      301 gcgactcccc tggcctttgc ccacaacccg tcccgggtgt gggagttcta ccactaccgg
      361 cgggaggtca tggggagcaa ggagcccaac gccgggcacc gcgccatagc cgagtgtgag
      421 acccggctgg gcaagcaggg ccggcgagtc gtggtcatca cccagaacat cgatgagctg
      481 caccgcaagg ctggcaccaa gaaccttctg gagatccatg gtagcttatt taaaactcga
      541 tgtacctctt gtggagttgt ggctgagaat tacaagagtc caatttgtcc agctttatca
      601 ggaaaaggtg ctccagaacc tggaactcaa gatgccagca tcccagttga gaaacttccc
      661 cggtgtgaag aggcaggctg cgggggcttg ctgcgacctc atgtcgtgtg gtttggagaa
      721 aacctggatc ctgccattct ggaggaggtt gacagagagc tcgcccactg tgatttatgt
      781 ctagtggtgg gcacttcctc tgtggtgtac ccagcagcca tgtttgcccc ccaggtggct
      841 gccaggggcg tgccagtggc tgaatttaac acggagacca ccccagctac gaacagattc
      901 aggtttcatt tccagggacc ctgtggaacg actcttcctg aagcccttgc ctgatgacga
      961 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1021 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1081 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1141 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1201 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1261 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1321 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1381 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1441 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1501 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1561 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1621 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1681 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1741 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1801 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1861 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1921 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1981 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2041 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2101 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2161 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2221 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2281 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2341 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2401 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2461 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     2521 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2761 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3181 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3241 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3301 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3361 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3421 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3481 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3541 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3601 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3661 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3721 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3781 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3841 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3901 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3961 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4021 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4081 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4141 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4201 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4261 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4321 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4381 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4441 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4501 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4561 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4621 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4681 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4741 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4801 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4861 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4921 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4981 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5041 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5101 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5161 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5221 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5281 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5341 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5401 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5461 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5521 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5581 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5641 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5701 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5761 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5821 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5881 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5941 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6001 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6061 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
