LOCUS       sequence-266312-        7210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Halotag-CNA35 peptide for Collagen labelling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7210)
  AUTHORS   Ju RJ, Falconer AD, Tang CKX, Dean KM, Fiolka RP, Sester DP, Nobis 
            M, Timpson P, Lomakin AJ, White MD, Oelz DB, Haass NK, Stehbens SJ
  TITLE     A Microtubule Mechanostat Enables Cells to Navigate Confined 
            Environments
  JOURNAL   bioRxiv 2022
REFERENCE   2  (bases 1 to 7210)
  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: "bioRxiv 
            2022"
COMMENT     Sequence Label: pET28a Halotag-CNA35
FEATURES             Location/Qualifiers
     source          1..7210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             157..1047
                     /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             1060..1080
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2075..2092
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2145..2163)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2159..2206
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2243..2698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2330..2349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2540..2561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2790..3605)
                     /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             complement(2790..3605)
                     /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     3513..3532
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3597..3616
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3727..4315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4216..4235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4469..4486
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4501..4640
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4566..4585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4726..4748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4742..4933)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5742..6824)
                     /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     6786..6805
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6825..6902)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7108..7127
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatgg cagaaatcgg tactggcttt
      181 ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt
      241 ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg
      301 cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt
      361 atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg
      421 gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc
      481 tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt
      541 atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc
      601 ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt
      661 atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat
      721 taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag
      781 ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg
      841 ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca
      901 ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc
      961 ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc
     1021 tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta ctttcagagc
     1081 tccggagaat tccacggatc cgcacgagat atttcatcaa cgaatgttac agatttaact
     1141 gtatcaccgt ctaagataga agatggtggt aaaacgacag taaaaatgac gttcgacgat
     1201 aaaaatggaa aaatacaaaa tggtgacatg attaaagtgg catggccgac aagcggtaca
     1261 gtaaagatag agggttatag taaaacagta ccattaactg ttaaaggtga acaggtgggt
     1321 caagcagtta ttacaccaga cggtgcaaca attacattca atgataaagt agaaaaatta
     1381 agtgatgttt cgggatttgc agaatttgaa gtacaaggaa gaaatttaac gcaaacaaat
     1441 acttcagatg acaaagtagc tacgataaca tctgggaata aatcaacgaa tgttacggtt
     1501 cataaaagtg aagcgggaac aagtagtgtt ttctattata aaacgggaga tatgctacca
     1561 gaagatacga cacatgtacg atggttttta aatattaaca atgaaaaaag ttatgtatcg
     1621 aaagatatta ctataaagga tcagattcaa ggtggacagc agttagattt aagcacatta
     1681 aacattaatg tgacaggtac acatagcaat tattatagtg gacaaagtgc aattactgat
     1741 tttgaaaaag cctttccagg ttctaaaata actgttgata atacgaagaa cacaattgat
     1801 gtaacaattc cacaaggcta tgggtcatat aatagttttt caattaacta caaaaccaaa
     1861 attacgaatg aacagcaaaa agagtttgtt aataattcac aagcttggta tcaagagcat
     1921 ggtaaggaag aagtgaacgg gaaatcattt aatcatactg tgcacaatat taatgctaat
     1981 gccggtattg aaggtactgt aaaaggtgaa ttaaaagttt taaaacagga taaagatacc
     2041 aaggcttcag tcgacctgta aggcattgct cgagcaccac caccaccacc actgagatcc
     2101 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2161 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2221 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2281 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2341 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2401 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2461 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2521 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2581 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2641 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2701 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2761 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2821 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2881 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2941 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3001 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3061 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3121 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3181 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3241 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3301 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3361 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3421 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3481 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3541 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3601 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3661 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3721 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3781 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3841 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3901 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3961 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4021 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4081 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4141 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4201 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4261 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4321 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4381 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4441 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4501 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4561 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4621 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4681 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4741 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4801 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4861 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4921 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4981 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5041 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5101 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5161 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5221 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5281 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5341 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5401 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5461 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5521 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5581 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5641 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5701 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5761 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5821 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5881 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5941 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6001 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6061 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6121 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6181 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6241 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6301 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6361 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6421 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6481 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6541 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6601 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6661 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6721 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6781 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6841 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6901 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6961 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     7021 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     7081 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     7141 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     7201 cccgcgaaat
//
