LOCUS       sequence-455390-        6367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""Empty"" N-terminal 6xHis-TEV-Halotag-HRV vector for easy DNA 
            Assembly for pulldowns (Bacterial Expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  TITLE     Molecular Toolbox
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6367)
  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            "Unpublished"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: MZB134
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(90..137)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     133..151
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(204..221)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(254..277)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(293..1183)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             complement(1184..1204)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1208..1225)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1239..1261
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1276..1300
                     /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(1300..1319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1301..1319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1403..1422)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1628..1705
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1706..2788
                     /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(1725..1744)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3597..3788
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3782..3804)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3890..4029
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3945..3964
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4044..4061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4215..4803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4295..4314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4974..5765,5766..5834))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5597..5616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5835..5939)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5965..6367,1..53))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6102..6123)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6314..6333
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
       61 gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
      121 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
      181 gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagtgc ggccgcaagc
      241 ttattattta aatcgggccc tgaaacagca cttccaggcc agtggttggc tcgccggaaa
      301 tctcgagcgt cgacagccag cgcgcgatct cgctgccgat caggtccggg ttgtcttctt
      361 gcagcagatt cagacccggg ccgatgtcca cagccttgca gttaggcagg cttttggcca
      421 ggcgagcggc ttcggccggt gggatcagaa cgcctggggt gccccagaac agcagcttcg
      481 ggacagggga ctggtgcagc cagtccatgt attcttcgac cagcgcgacg atgttcgctg
      541 gctcaccggc gattggcagc tcgtttggga agcgccacag tggctcgcgg tcaacaggat
      601 tcaggaacgg ctcgcggtaa tggtccatct cgacttcagt cagcgggcgg acgacaccca
      661 tcggcagcgt accctcgata aaaacgttct gatcgatgat cagcttgcgg ccgacgtcgg
      721 tggtgcggaa ggcctggaag gtctcgcggg caaattctgg ccattcgtcc caggtcggga
      781 tagggcggat gaactccata aatgcaatac ctttgacgcg ctctggattg cgcttggccc
      841 agtggaaacc cagagcggag ccccagtcgt gaatgaccag gacgacctct tccagaccca
      901 gggcttcgat gaaggcatcc atgaagcgga cgtggtcgtc gaagaaataa cccaggtctg
      961 gtttgtcgga tttgcccata ccgatcaggt ctggagcaat gcagcgatgg gtcggtgcaa
     1021 catgcgggat gatgttgcgc cacacgtagg aggaggtcgg gttaccgtgc aggaacagca
     1081 caggggtgcc atcgcgcgga ccaacatcga cgtagtgcat gcgctcgccc aggacttcca
     1141 cataatgggg gtcgaatgga aagccagtac cgatttctgc catgccctgg aagtacaggt
     1201 tctcgccgtg gtgatgatgg tgatggccca tatgtatatc tccttcttaa agttaaacaa
     1261 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
     1321 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1381 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1441 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1501 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1561 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1621 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1681 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1741 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1801 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1861 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1921 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1981 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     2041 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     2101 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2161 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2221 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2281 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2341 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2401 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2461 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     2521 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2581 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2641 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2701 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2761 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     2821 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2881 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     2941 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     3001 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     3061 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3121 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3181 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3241 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3301 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3361 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3421 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3481 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3541 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3601 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     3661 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3721 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3781 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3841 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3901 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     3961 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4021 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4081 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4141 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4201 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4261 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4321 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4381 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4441 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4501 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4561 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4621 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4681 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4741 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4801 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4861 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4921 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4981 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5041 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5101 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5161 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5221 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5281 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5341 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5401 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5461 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5521 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5581 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5641 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5701 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5761 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5821 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5881 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5941 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6001 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6061 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6121 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6181 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6241 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6301 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6361 cggtcac
//
