LOCUS       sequence-404735-        7188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For SnoopLigase2 expression within secretory pathway of mammalian 
            cells. N775Q mutation to remove glycosylation site, KDEL to retain 
            in Endoplasmic Reticulum, MBP for improved solubility.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7188)
  AUTHORS   Keeble AH, Wood DP, Howarth M
  TITLE     Design and Evolution of Enhanced Peptide-Peptide Ligation for 
            Modular Transglutaminase Assembly.
  JOURNAL   Bioconjug Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122.
  PUBMED    37289810
REFERENCE   2  (bases 1 to 7188)
  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: "Bioconjug 
            Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122."
COMMENT     Sequence Label: pcDNA3.1 HA-MBP-SnoopLigase2 N775Q-KDEL
FEATURES             Location/Qualifiers
     source          1..7188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    203..324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(240..259)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     294..313
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(373..389)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(373..389)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(386..408)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    397..413
                     /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)."
     promoter        complement(join(421..427,428..445,446..451))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    466..487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(604..621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(775..1363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(855..874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1534..2325,2326..2394))
                     /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     2157..2176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2395..2499)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2574..2593)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2765..3144
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3145..3348
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3299..3319
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3393..3412
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3393..3411
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3540..3566
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3582..4679
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4653..4676
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4710..4727
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5309..5326)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5315..5539
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5585..6013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5672..5691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5882..5903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6022..6042)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6027..6356
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6207..6342
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6269..6288
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(6423..7188,1..29)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6477..6496)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7087..7106
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
       61 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
      121 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
      181 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
      241 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
      301 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
      361 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
      421 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
      481 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
      541 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
      601 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      661 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      721 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      781 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      841 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      901 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      961 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1021 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1081 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1141 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1201 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1261 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1321 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1381 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1441 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1501 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1561 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1621 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1681 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1741 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1801 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1861 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1921 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1981 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2041 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2101 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2161 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2221 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2281 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2341 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2401 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2461 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2521 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     2581 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     2641 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     2701 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     2761 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2821 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2881 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2941 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3001 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3061 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3121 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     3181 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     3241 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3301 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     3361 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     3421 gctggctagc gtttaaacgg gccctctaga ctcgagcggc cgccaccatg aacacccaga
     3481 tcctggtgtt cgccctgatc gccatcattc ccaccaacgc cgacaagatc ggatctggct
     3541 acccatacga tgttccagat tacgctagtg gagaaagtgg taaaatcgaa gaaggtaaac
     3601 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
     3661 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
     3721 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
     3781 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
     3841 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
     3901 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
     3961 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
     4021 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
     4081 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
     4141 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
     4201 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
     4261 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
     4321 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
     4381 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     4441 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     4501 acgaggaaga gttggcgaaa gatccacgta ttgccgccac tatggaaaac gcccagaaag
     4561 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     4621 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
     4681 attcgagctc gggtagcagt ggtgcagcac atcatcatca ccatcatagc tctggtggta
     4741 gtggtgtgaa taagaacgat aaaaagccgc tgcgtggtgc cgtgtttagc ctgcagaaac
     4801 agcatcccga ctatcccgat atctatggcg cgattgatca gcaggggacc tatcaaaatg
     4861 tgcgtaccgg cgaagatggt aaactgacct ttaagaatct gagcgatggc aaatatcgcc
     4921 tgattgaaaa tagcgaaccc ccgggctata aaccggtgca gaataagccg attgtgagct
     4981 ttcgtattgt ggatggcgaa gtgcgtgatg tgaccagcat tgtgccgccg ggtgtgccgg
     5041 ctacatatga atttaccgga ggaggtggct ctaaagatga actgtagtag accggtaagc
     5101 tgagcagcgg ctacaaggac gtgatcctgt ggttcagctt cggcgccagc tgcttcatcc
     5161 tgctggccat cgtgatgggc ctggtgttca tctgcgtgaa gaacggcaac atgcggtgca
     5221 ccatctgcat ctgatgatat cgaattccac cacactggac tagtggatcc gagctcggta
     5281 ccaagcttaa gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5341 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5401 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5461 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     5521 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     5581 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5641 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5701 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5761 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5821 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5881 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5941 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6001 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     6061 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     6121 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     6181 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     6241 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     6301 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     6361 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     6421 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     6481 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     6541 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     6601 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     6661 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     6721 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     6781 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     6841 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     6901 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     6961 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     7021 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     7081 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     7141 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgcct
//
