LOCUS       sequence-147900-        4992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyTagged GlpG protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4992)
  AUTHORS   Min D, Arbing MA, Jefferson RE, Bowie JU
  TITLE     A simple DNA handle attachment method for single molecule mechanical
            manipulation experiments.
  JOURNAL   Protein Sci. 2016 Aug;25(8):1535-44. doi: 10.1002/pro.2952. Epub 
            2016 Jun 6.
  PUBMED    27222403
REFERENCE   2  (bases 1 to 4992)
  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"; doi: "10.1002/pro.2952"; 
            journalName: "Protein Sci"; date: "2016-08"; volume: "25"; issue: 
            "8"; pages: "1535-44"
COMMENT     Sequence Label: pSpyTag-GlpG
FEATURES             Location/Qualifiers
     source          1..4992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(192..197,198..214,215..221)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    229..245
                     /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     234..256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    262..331
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             382..408
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             418..435
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             442..480
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             1093..1131
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     primer_bind     complement(1174..1191)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1344..1430
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1522..1549
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1569..1660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1661..1729,1730..2521)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1879..1898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2692..3280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3181..3200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3434..3451
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3466..3606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3692..3714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3792..3869
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3870..4952
                     /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(3889..3908)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg
       61 gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
      121 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
      181 gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt gtgagcggat
      241 aacaatttca cacaggaaac agcgccgctg agaaaaagcg aagcggcact gctctttaac
      301 aatttatcag acaatctgtg tgggcactcg accggaatta tcgattaact ttattattaa
      361 aaattaaaga ggtatatatt aatgtatcga ttaaataagg aggaataaac catggggcat
      421 catcatcatc atcatgagct cgctcatatt gtaatggtcg atgcgtataa accgacgaaa
      481 ggttcaggag agtcaggcgc cgcctgtttg cgtgaacgcg caggtccggt aacctgggtg
      541 atgatgatcg ccgccgtggt ggtgtttatt gccatgcaaa ttctcggcga tcaggaagtg
      601 atgttatggc tggcctggcc attcgatcca acactgaaat ttgagttctg gcgttacttc
      661 acccacgcgt taatgcactt ctcgctgatg catatcctct ttaacctgct ctggtggtgg
      721 tatctcggcg gtgcggtgga aaaacgcctc ggtagcggta agctaattgt cattacgctt
      781 atcagcgccc tgttaagcgg ctatgtgcag caaaaattca gcgggccgtg gtttggcggg
      841 ctttctggcg tggtgtatgc gctgatgggc tacgtctggc tacgtggcga acgcgatccg
      901 caaagtggca tttacctgca acgtgggtta attatctttg cgctgatctg gattgtcgcc
      961 ggatggtttg atttgtttgg gatgtcgatg gcgaacggag cacacatcgc cgggttagcc
     1021 gtgggtttag cgatggcttt tgttgattcg ctcaatgcgc gaaaacgaaa atgtggtagt
     1081 ggtgaaagtg gtgcccacat cgtgatggtg gacgcctaca agccgacgaa ggagtaagct
     1141 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     1201 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     1261 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
     1321 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     1381 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     1441 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     1501 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     1561 aaactctttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2341 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2821 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3481 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3601 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3721 ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat
     3781 gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc
     3841 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     3901 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     3961 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4021 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4081 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4141 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4201 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4261 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4321 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4381 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4441 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4501 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4561 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     4621 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     4681 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     4741 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     4801 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     4861 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4921 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagcgc
     4981 gaattgatct gg
//
