LOCUS       sequence-197574-        6099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6099)
  AUTHORS   Bennett MJ, Huey-Tubman KE, Herr AB, West AP, Ross SA, Bjorkman PJ
  TITLE     Inaugural Article: A linear lattice model for polyglutamine in 
            CAG-expansion diseases.
  JOURNAL   Proc Natl Acad Sci U S A. 2002 Sep 3. 99(18):11634-9.
  PUBMED    12193654
REFERENCE   2  (bases 1 to 6099)
  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: "Proc Natl 
            Acad Sci U S A. 2002 Sep 3. 99(18):11634-9."
COMMENT     Sequence Label: pET32a-HD25Q
FEATURES             Location/Qualifiers
     source          1..6099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(206..223)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(503..517)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(569..895)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             903..925
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    940..964
                     /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)."
     primer_bind     complement(964..983)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(965..983)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1071..1090)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1296..1373
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1374..2456
                     /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     complement(1393..1412)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3265..3456
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3450..3472)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3558..3697
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3613..3632
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3712..3729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3883..4471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3963..3982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4642..5433,5434..5502))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5265..5284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5503..5607)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5633..6088)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5770..5791)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5982..6001
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcctaatgat gatgatgatg atgaggtcgg tgcagaggct
      241 cctcagccac agctgggcct ggaggaggtg gaggtggagg tggaggaggt tgtggctgag
      301 gcagcagagg ctgtgcctgt ggaggaggtt gaggaagttg aggaggtggg ggtggagggg
      361 gaggtggtgg cggttgttgc tgttgctgct gttgttgctg ttgctgctgt tgttgctgtt
      421 gctgctgttg ttgctgttgc tgctgttgga agcttttgag ggactcgaag gccttcatca
      481 gcttttccag ggtcgccatg gccttgtcgt cgtcgtcggt acccagatct gggctgtcca
      541 tgtgctggcg ttcgccagaa ccagaaccgg ccaggttagc gtcgaggaac tctttcaact
      601 gacctttaga cagtgcaccc actttggttg ccgccacttc accgtttttg aacagcagca
      661 gagtcgggat accacggatg ccatatttcg gcgcagtgcc agggttttga tcgatgttca
      721 gttttgcaac ggtcagtttg ccctgatatt cgtcagcgat ttcatccaga atcggggcga
      781 tcattttgca cggaccgcac cactctgccc agaaatcgac gaggatcgcc ccgtccgctt
      841 tgagtacatc cgtgtcaaaa ctgtcgtcag tcaggtgaat aattttatcg ctcatatgta
      901 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
      961 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tcgatctcga tcctctacgc
     1021 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     1081 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     1141 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
     1201 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     1261 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     1321 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     1381 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     1441 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     1501 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     1561 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     1621 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     1681 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     1741 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     1801 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     1861 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     1921 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     1981 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     2041 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     2101 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     2161 gcgttggtgc ggacatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     2221 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     2281 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     2341 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     2401 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     2461 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     2521 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     2581 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     2641 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     2701 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     2761 ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
     2821 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     2881 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     2941 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     3001 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     3061 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     3121 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     3181 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     3241 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     3301 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     3361 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     3421 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3481 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3541 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     3601 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat
     3661 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     3721 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3781 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3841 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3901 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3961 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4021 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4081 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4141 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4201 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4261 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4321 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4381 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4441 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4501 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4561 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4621 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4681 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4741 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4801 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4861 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4921 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     4981 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5041 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5101 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5161 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5221 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5281 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5341 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5401 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5461 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5521 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5581 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5641 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5701 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5761 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5821 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5881 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5941 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6001 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6061 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
