LOCUS       sequence-351934-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EndoH in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  TITLE     Bacterial expression of MBP-His8-EndoH
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7480)
  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            "Unpublished"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: pHN764
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(718..723,724..739,740..746)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    754..770
                     /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     759..781
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             840..1940
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1797..1806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1914..1937
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1989..2000
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2010..2033
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2885..2902)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2885..2901)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2894..2916)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3080..3097)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3250..3336
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3428..3455
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3474..3565
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3566..3634,3635..4426)
                     /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(3784..3803)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4468..4981)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4663..4684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4875..4894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5092..5680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5581..5600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5834..5851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5866..6006
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5932..5951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6092..6114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6108..6299)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6795..6872
                     /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             join(6873..7480,1..475)
                     /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(6892..6911)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
       61 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
      121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
      181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
      241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
      301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
      361 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg acagcttatc
      541 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
      601 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
      661 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
      721 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
      781 gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga ccatagatta
      841 tgaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
      901 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
      961 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1021 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1081 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1141 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1201 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1261 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     1321 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     1381 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     1441 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     1501 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     1561 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     1621 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     1681 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     1741 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     1801 ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     1861 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     1921 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     1981 acctcgggat cgagggaagg ggaggatccc accaccacca ccatcatcat catgcaccag
     2041 ctccggtaaa gcagggtcca acgtcggtgg catacgtgga ggtcaataac aacagcatgc
     2101 ttaatgttgg aaaatatact cttgccgacg gaggtggcaa tgcctttgat gtagcagtaa
     2161 ttttcgcggc aaatatcaac tatgatacgg gcactaaaac cgcctattta catttcaatg
     2221 aaaacgtcca acgcgtactg gataacgctg ttactcagat ccgcccgctg caacagcaag
     2281 ggatcaaggt cctgttaagc gtgcttggta accatcaagg tgcggggttc gctaacttcc
     2341 cttctcagca agcggcatct gcgtttgcca aacagctgag tgatgcggta gctaaatatg
     2401 gcctggatgg tgtagacttt gacgatgaat atgccgagta tggcaacaac gggaccgccc
     2461 aacctaatga ttcgtctttt gtgcacctgg tgaccgccct gcgcgctaat atgccagata
     2521 aaattattag tctctacaat atcggcccgg cggcctcccg cctgagttac ggcggggtgg
     2581 acgtgtcgga taaatttgat tatgcctgga acccctatta cggtacctgg caagtgccag
     2641 gaattgcgct gcccaaagcg caattgagcc cggccgccgt cgaaattggc cgtacctcac
     2701 gtagcacagt cgctgacctg gcgcgccgta cagtcgatga aggctatggc gtgtacttga
     2761 cctacaatct ggacggcggt gaccgcacgg cggacgttag cgcctttacc cgcgaattat
     2821 acggttcaga agcggttcgt acgccatgac tcgagtctag agtcgacctg caggcaagct
     2881 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2941 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     3001 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcagcttggc tgttttggcg
     3061 gatgagataa gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa
     3121 aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag
     3181 aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact
     3241 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     3301 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     3361 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
     3421 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt
     3481 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3541 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3601 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3661 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3721 taagatcctt gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt
     3781 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3841 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3901 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3961 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4021 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4081 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4141 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     4201 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4261 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4321 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4381 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4441 ttactcatat atactttaga ttgatttacc ccggttgata atcagaaaag ccccaaaaac
     4501 aggaagattg tataagcaaa tatttaaatt gtaaacgtta atattttgtt aaaattcgcg
     4561 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4621 tataaatcaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4681 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     4741 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
     4801 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4861 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4921 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4981 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5041 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5101 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5161 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5221 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5281 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5341 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5401 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5461 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5521 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5581 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5641 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5701 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5761 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5821 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     5881 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     5941 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     6001 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6061 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6121 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgcagc
     6181 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     6241 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     6301 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     6361 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     6421 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     6481 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     6541 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     6601 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     6661 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     6721 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     6781 ctgcccgaaa ttccgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc
     6841 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     6901 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     6961 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     7021 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     7081 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     7141 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     7201 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     7261 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     7321 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     7381 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     7441 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
//
