LOCUS       sequence-343760-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag11 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  AUTHORS   Frei MS, Tarnawski M, Roberti MJ, Koch B, Hiblot J, Johnsson K
  TITLE     Engineered HaloTag variants for fluorescence lifetime multiplexing.
  JOURNAL   Nat Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x.
  PUBMED    34916672
REFERENCE   2  (bases 1 to 5992)
  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: "Nat 
            Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x."
COMMENT     Sequence Label: pET-51b-HaloTag11
FEATURES             Location/Qualifiers
     source          1..5992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..49
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      86..541
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(173..192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     383..404
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(656..1447,1448..1516))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1279..1298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1517..1609)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1690..2278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2179..2198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2432..2449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2464..2603
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2529..2548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2689..2711
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2705..2896)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3468..4550)
                     /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     4512..4531
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4551..4628)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4834..4853
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4941..4960
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4941..4959
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4960..4984
                     /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             4999..5021
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5031..5060
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5061..5081
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(join(5980..5992,1..6))
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
       61 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      121 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      181 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      241 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      301 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      361 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
      421 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
      481 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
      541 tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
      601 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
      661 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
      721 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      781 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
      841 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      901 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      961 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1021 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1081 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1141 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1201 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1261 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1321 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1381 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     1441 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     1501 aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat
     1561 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac
     1621 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1681 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1741 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1801 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1861 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1921 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1981 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2041 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2101 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2161 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2221 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2281 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2341 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2401 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2461 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     2521 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     2581 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     2641 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2701 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     2761 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2821 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2881 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     2941 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3001 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     3061 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     3121 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     3181 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     3241 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     3301 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca
     3361 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3421 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3481 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3541 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3601 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3661 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3721 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     3781 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     3841 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     3901 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     3961 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4021 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4081 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4141 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4201 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4261 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4321 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4381 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4441 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4501 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4561 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4621 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4681 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4741 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4801 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     4861 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     4921 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     4981 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg catcaccacc
     5041 atcatcacca tcaccaccac gaaaacctgt attttcaggg catcggtact ggctttccat
     5101 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
     5161 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
     5221 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
     5281 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
     5341 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
     5401 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
     5461 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
     5521 aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     5581 agggtacgct gccgtggggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     5641 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     5701 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     5761 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     5821 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     5881 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     5941 tgtctactct ggagatttaa ttaacctagg ctgctgccac cgctgagcaa ta
//
