LOCUS       sequence-457073-        5958 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For expression of ULP1 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5958)
  TITLE     LiWang lab plasmids
REFERENCE   2  (bases 1 to 5958)
  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: pET28b-ULP1-405-621
FEATURES             Location/Qualifiers
     source          1..5958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(23..70)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     66..84
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(137..154)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(833..850)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(860..877)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             896..918
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    933..957
                     /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(957..976)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(958..976)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1060..1079)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1285..1362
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1363..2445
                     /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(1382..1401)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3254..3445
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3439..3461)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3547..3686
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3602..3621
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3701..3718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3872..4460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3952..3971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4571..4590)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4582..5397
                     /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             4582..5397
                     /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     complement(4655..4674)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5489..5944)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5626..5647)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5838..5857
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
       61 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
      121 tagcagccgg atctcagtgg tggtggtggt ggtgctcgag ctattttaaa gcgtcggtta
      181 aaatcaaatg ggcaataaat cttctcatcc taatcgcatc tttataatca aaatccaatg
      241 gcgcatctgc acttccatag agagtattca tacaaacata tattccacag tcgtagccat
      301 ttggttgctg cggacaatct aaatgaatca aatcaaagtc ttctcctatt gtatgcttac
      361 tttcctccat aacatatttt tgcaagtcag tcagtatagc gaaactcata gcatttggac
      421 cattcgataa tgaatctacg taacctatag ttttcttttt taaatcaatt atgcccaacg
      481 cccagtggga ttggttcaaa tttattggtg taaagatttt atcaagttta tcaatttgtg
      541 tcttctttct cttcatccac ctccggacgc cttgataacc cctttctgat aaattggtat
      601 agaaaaatga attaaacgcc actgtattag gggtagattt ttcaatgtat ttcataaaaa
      661 actcaatgat agtgtcattt agccatcttc gtggtgccaa ggtcttaaaa tcacgtactg
      721 ttatctctat attatctcta ttcattaact gagtattttc tctagatgcc aaagcttttt
      781 gtacttggtc atcgtctttt tcatttaatt caggaacaag gctagccata tggctgccgc
      841 gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcccatg gtatatctcc
      901 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      961 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
     1021 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     1081 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     1141 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     1201 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     1261 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     1321 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     1381 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     1441 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     1501 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     1561 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     1621 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     1681 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     1741 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     1801 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     1861 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1921 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1981 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     2041 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     2101 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     2161 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     2221 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     2281 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     2341 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     2401 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2461 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     2521 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     2581 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     2641 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     2701 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     2761 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2821 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2881 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2941 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3001 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3061 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3121 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3181 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     3241 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     3301 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3361 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3421 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3481 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3541 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3601 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     3661 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     3721 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3781 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3841 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3901 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3961 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4021 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4081 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4141 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4201 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4261 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4321 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4381 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4441 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4501 ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata
     4561 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg
     4621 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     4681 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     4741 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     4801 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     4861 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     4921 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     4981 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     5041 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     5101 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     5161 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     5221 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     5281 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     5341 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa
     5401 ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5461 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     5521 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5581 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5641 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5701 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5761 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5821 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5881 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5941 gcgtcccatt cgccaatc
//
