LOCUS       sequence-381376-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal (His)6-TEV cleavage site fusion of human gene 
            or portion of gene, with point mutation, in bacterial strains. pET 
            based vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6229)
  TITLE     Davis Lab plasmids
REFERENCE   2  (bases 1 to 6229)
  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: PRPF4.001.169.S34E
FEATURES             Location/Qualifiers
     source          1..6229
                     /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             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(730..748)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      744..791
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(966..971,972..988,989..994))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1021..1039)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1107..1211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1212..1280,1281..2072)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1430..1449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2243..2831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2732..2751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2985..3002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3017..3156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3082..3101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3242..3264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3258..3449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4761..5843)
                     /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     5805..5824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5844..5921)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6127..6146
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga
      121 gaaaacttgt atttccaggg catggcttcc tcgcgagcct cttccacgca ggcaaccaaa
      181 actaaagcac ccgacgactt agttgctccg gtcgtgaaga aaccacacat ctattatgga
      241 gagttggaag agaaggagag ggagcgtctg gccaaaggag agtctgggat tttggggaaa
      301 gacggactta aagcagggat cgaagctgga aatattaata taacctctgg agaagtgttt
      361 gaaattgaag agcatatcag cgagcgacag gcagaagtat tggctgagtt tgagagaagg
      421 aagcgagccc ggcagatcaa tgtttccaca gatgactcag aggtcaaagc ttgccttaga
      481 gccttggggg aacccatcac actttttgga gagggtcctg ctgaaagaag agaaaggtta
      541 agaaatatcc tctcagttgt cggtactgat gccttgaaaa agaccaaaaa ggatgatgag
      601 aagtctaaaa agtccaaaga agagtatcag caaacctggt atcatgaata gtgatgacga
      661 agcttgcggc cgcactcgag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      721 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      781 ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac
      841 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
      901 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
      961 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
     1021 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     1081 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1141 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1201 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1261 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1321 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1381 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1441 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1501 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1561 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1621 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1681 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1741 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1801 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1861 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1921 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1981 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2041 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2101 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2161 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2221 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2281 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2341 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2401 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2461 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2521 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2581 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2641 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2701 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2761 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2821 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2881 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2941 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3001 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3061 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3121 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3181 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3241 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3301 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3361 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3421 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3481 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3541 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3601 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3661 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcggggcgct
     3721 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3781 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3841 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3901 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     3961 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     4021 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     4081 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     4141 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     4201 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
     4261 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     4321 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     4381 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     4441 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4501 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4561 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4621 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4681 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4741 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4801 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4861 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4921 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4981 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5041 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5101 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5161 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5221 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5281 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5341 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5401 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5461 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5521 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5581 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5641 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5701 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5761 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5821 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5881 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5941 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6001 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6061 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6121 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6181 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
