LOCUS       sequence-471362-        6465 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 6465)
  TITLE     SGC+year
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6465)
  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: 
            "unpublished"
COMMENT     Sequence Label: HAO1
FEATURES             Location/Qualifiers
     source          1..6465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     82..101
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     185..204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        185..203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    204..228
                     /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             243..265
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             276..293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..338
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1514..1531
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1584..1602)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1598..1645
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1682..2137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1769..1788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1979..2000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2229..3044)
                     /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(2229..3044)
                     /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     2952..2971
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3036..3055
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3166..3754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3655..3674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3908..3925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3940..4079
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4005..4024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4165..4187
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4181..4372)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5181..6263)
                     /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     6225..6244
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6264..6341)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
       61 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
      121 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
      181 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
      241 attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat cattcttctg
      301 gtgtagatct gggtaccgag aacctgtact tccaatccat gctcccccgg ctaatttgta
      361 tcaatgatta tgaacaacat gctaaatcag tacttccaaa gtctatatat gactattaca
      421 ggtctggggc aaatgatgaa gaaactttgg ctgataatat tgcagcattt tccagatgga
      481 agctgtatcc aaggatgctc cggaatgttg ctgaaacaga tctgtcgact tctgttttag
      541 gacagagggt cagcatgcca atatgtgtgg gggctacggc catgcagcgc atggctcatg
      601 tggacggcga gcttgccact gtgagagcct gtcagtccct gggaacgggc atgatgttga
      661 gttccwgggc cacctcctca attgaagaag tggcggaagc tggtcctgag gcacttcgtt
      721 ggctgcaact gtatatctac aaggaccgag aagtcaccaa gaagctagtg cggcaggcag
      781 agaagatggg ctacaaggcc atatttgtga cagtggacac accttacctg ggcaaccgtc
      841 tggatgatgt gcgtaacaga ttcaaactgc cgccacaact caggatgaaa aattttgaaa
      901 ccagtacttt atcattttct cctgaggaaa attttggaga cgacagtgga cttgctgcat
      961 atgtggctaa agcaatagac ccatctatca gctgggaaga tatcaaatgg ctgagaagac
     1021 tgacatcatt gccaattgtt gcaaagggca ttttgagagg tgatgatgcc agggaggctg
     1081 ttaaacatgg cttgaatggg atcttggtgt cgaatcatgg ggctcgacaa ctcgatgggg
     1141 tgccagccac tattgatgtt ctgccagaaa ttgtggaggc tgtggaaggg aaggtggaag
     1201 tcttcctgga cgggggtgtg cggaaaggca ctgatgttct gaaagctctg gctcttggcg
     1261 ccaaggctgt gtttgtgggg agaccaatcg tttggggctt agctttccag ggggagaaag
     1321 gtgttcaaga tgtcctcgag atactaaagg aagaattccg gttggccatg gctctgagtg
     1381 ggtgccagaa tgtgaaagtc atcgacaaga cattggtgag gaaaaatcct ttggccgttt
     1441 ccaagatctg acagtaaagg tggatacgga tccgaattcg agctccgtcg acaagcttgc
     1501 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1561 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1621 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1681 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1741 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1801 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1861 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1921 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1981 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2041 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2101 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2161 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2221 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2281 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2341 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2401 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2461 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2521 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2581 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2641 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2701 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2761 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2821 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2881 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2941 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3001 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3061 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     3121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3301 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3901 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3961 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4021 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4081 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4141 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4201 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4261 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4321 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4381 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4441 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4501 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4561 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4621 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4681 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4741 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4801 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4861 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4921 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4981 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5041 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5101 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5161 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5281 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5341 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5401 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5461 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5521 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5581 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5641 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5701 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5761 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5821 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5881 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5941 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6001 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6061 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6121 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6181 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6241 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6301 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6361 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6421 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcc
//
