LOCUS       sequence-457332-        5436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""Empty"" N-terminal TEV cleavable 6x His tag vector for rapid 
            cloning/DNA Assembly via SmaI restriction site (Bacterial 
            Expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5436)
  TITLE     Molecular Toolbox
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5436)
  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            "Unpublished"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: MZB133
FEATURES             Location/Qualifiers
     source          1..5436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             351..542
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(536..558)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    644..783
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     699..718
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(798..815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(969..1557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1049..1068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1728..2519,2520..2588))
                     /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     2351..2370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2589..2693)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2719..3174)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2856..2877)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3068..3087
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3211..3258)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3254..3272
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3325..3342)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3374..3394)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(3398..3415)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3429..3451
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3466..3490
                     /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(3490..3509)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3491..3509)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3593..3612)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3818..3895
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3896..4978
                     /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(3915..3934)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
       61 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
      121 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
      181 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
      241 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
      301 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
      361 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
      421 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
      481 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
      541 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
      601 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
      661 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
      721 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
      781 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      961 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1021 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1141 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1381 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     1441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     1741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2041 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2101 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     2281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     2341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     2401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     2461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     2521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2701 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2761 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2821 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2881 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2941 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3001 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3061 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3121 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     3181 cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc
     3241 cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg
     3301 ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgctcgagtg cggccgcaag
     3361 cttattaccc ggggccctgg aagtacaggt tctcgccgtg gtgatgatgg tgatggccca
     3421 tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc
     3481 tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac
     3541 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     3601 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     3661 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     3721 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     3781 atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac
     3841 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     3901 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     3961 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     4021 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     4081 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     4141 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     4201 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     4261 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     4321 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     4381 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     4441 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     4501 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     4561 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     4621 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     4681 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     4741 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     4801 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     4861 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     4921 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4981 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     5041 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     5101 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     5161 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     5221 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     5281 tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc
     5341 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat
     5401 caccgatacg cgagcgaacg tgaagcgact gctgct
//
