LOCUS       sequence-381379-        6212 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses N-terminal (His)6-Thrombin 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 6212)
  TITLE     Davis Lab plasmids
REFERENCE   2  (bases 1 to 6212)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: ppie.001.301.W257A
FEATURES             Location/Qualifiers
     source          1..6212
                     /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             1077..1094
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1147..1165)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1161..1208
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1245..1700
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1332..1351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1542..1563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1792..2607)
                     /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(1792..2607)
                     /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     2515..2534
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2599..2618
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2729..3317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3218..3237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3471..3488
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3503..3642
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3568..3587)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3728..3750
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3744..3935)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4744..5826)
                     /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     5788..5807
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5827..5904)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6110..6129
                     /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 tagtatggcc accaccaagc gcgtcttgta cgtgggtgga
      181 ctggcagagg aagtggacga caaagttctt catgctgcgt tcattccttt tggagacatc
      241 acagatattc agattcctct ggattatgaa acagaaaagc accgaggatt tgcttttgtt
      301 gaatttgagt tggcagagga tgctgcagca gctatcgaca acatgaatga atctgagctt
      361 tttggacgta caattcgtgt caatttggcc aaaccaatga gaattaagga aggctcttcc
      421 aggccagttt ggtcagatga tgactggttg aagaagtttt ctgggaagac gcttgaagag
      481 aataaagagg aagaagggtc agagcctccc aaagcagaga cccaggaggg agagcccatt
      541 gctaaaaagg cccgctcaaa tcctcaggtg tacatggaca tcaagattgg gaacaagccg
      601 gctggccgca tccagatgct cctgcgttct gatgtcgtgc ccatgacagc agagaatttc
      661 cgctgcctgt gcactcatga aaagggcttt ggctttaagg gaagcagctt ccaccgcatc
      721 atcccccagt tcatgtgcca gggcggtgat ttcacaaacc acaatggcac tgggggcaag
      781 tccatctatg ggaagaagtt cgatgatgaa aactttatcc tcaagcatac gggaccaggt
      841 ctactatcca tggccaactc tggcccaaac accaatggct ctcagttctt cctgacatgt
      901 gacaagacag acgcgctgga tggcaagcat gtggtgtttg gagaggtcac cgaaggccta
      961 gatgtcttgc ggcaaattga ggcccagggc agcaaggacg ggaagccaaa gcagaaggtg
     1021 atcatcgccg actgtgggga gtacgtgtga tgacgaagct tgcggccgca ctcgagcacc
     1081 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1141 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1201 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1261 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1321 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1381 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1441 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1501 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1561 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1621 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1681 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1741 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1801 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1861 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1921 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1981 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2041 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2101 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2161 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2221 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2281 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2341 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2401 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2461 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2521 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2581 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2641 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2701 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2761 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2821 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2881 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2941 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3001 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3061 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3361 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3421 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3481 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3541 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3601 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3661 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3721 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3781 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3841 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3901 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3961 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4021 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4081 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4141 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4201 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4261 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4321 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4381 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4441 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4501 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4561 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4621 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4681 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4741 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4801 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4861 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4921 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4981 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5041 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5101 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5161 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5221 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5281 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5341 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5401 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5461 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5521 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5581 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5641 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5701 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5761 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5821 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5881 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5941 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6001 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6061 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6121 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6181 cgtagaggat cgagatctcg atcccgcgaa at
//
