LOCUS       sequence-455392-        5249 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Empty C-terminal 8xHis tag vector for rapid cloning/DNA assembly via
            SwaI restriction site for E. coli based protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5249)
  TITLE     Molecular Toolbox
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5249)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: MZB223
FEATURES             Location/Qualifiers
     source          1..5249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(24..479)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(161..182)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     373..392
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(516..563)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     559..577
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(634..657)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             676..698
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    717..733
                     /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(737..756)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(738..756)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(844..863)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1069..1146
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1147..2229
                     /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(1166..1185)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3038..3229
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3223..3245)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3331..3470
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3386..3405
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3485..3502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3656..4244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3736..3755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4355..4374)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4366..5181
                     /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             4366..5181
                     /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(4439..4458)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
       61 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
      121 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
      181 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
      241 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
      301 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
      361 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
      421 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
      481 ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
      541 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
      601 tttcgggctt tgttagcagc cggatcctta ttaatgatga tgatggtggt ggtgatgatt
      661 taaatcatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
      721 tatccgctca caatttccct atagtgagtc gtattaattt cgcgggatcg agatcgatct
      781 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
      841 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
      901 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
      961 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     1021 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
     1081 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aatagagtca attcagggtg
     1141 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     1201 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     1261 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     1321 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     1381 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     1441 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     1501 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     1561 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     1621 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     1681 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     1741 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     1801 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     1861 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     1921 gagtccgggc tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac
     1981 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     2041 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     2101 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     2161 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2221 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     2281 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     2341 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     2401 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     2461 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     2521 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
     2581 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     2641 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     2701 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     2761 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     2821 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     2881 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     2941 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     3001 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     3061 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     3121 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     3181 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3241 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3301 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3361 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     3421 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3481 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3541 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3601 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3661 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3721 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3781 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3841 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3901 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3961 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4021 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4081 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4141 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4201 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4261 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4321 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     4381 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
     4441 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     4501 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     4561 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     4621 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     4681 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     4741 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     4801 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     4861 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     4921 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     4981 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     5041 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     5101 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     5161 atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta
     5221 gaaaaataaa caaatagggg ttccgcgca
//
