LOCUS       sequence-492806-        7011 bp    DNA     circular SYN 14-NOV-2025
DEFINITION  Maltose binding protein N-terminally fused to His-tagged N-terminal 
            LCGC14 PolB2split intein fragment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  TITLE     Three-Segment Protein Labeling Using a New Highly Efficient and 
            Cysteine-Less Split Intein Identified with Computational Prediction 
            of Aggregation Properties
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7011)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pMAL-c2X_MBP-LcgcN(C24S)-H6
FEATURES             Location/Qualifiers
     source          1..7011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /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             81..1163
                     /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(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /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
                     IAYPIAIEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2485..2494
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3076..3093
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3104..3121)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3104..3120)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3113..3135)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3299..3316)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3469..3555
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3647..3674
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3693..3784
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3785..3853,3854..4645)
                     /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(4003..4022)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4687..5200)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4882..4903)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5094..5113
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5311..5899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5800..5819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6053..6070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6085..6225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6151..6170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6311..6333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6327..6518)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagattatg aaaactgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctatt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcagaattc
     2701 gataccgata gcgtggacgc tgatacaatt attaagacga actacggaga gatgactatc
     2761 gaaaatttgt ttaagtctag tagcattaaa ggtccgtcct gggcaatcga cgatcaggaa
     2821 tttaccattt atgaccagat ccaaattttg acttacgacc caaagacgaa cgaggaaatt
     2881 taccgtccct tcgaatatgt atatcgtcat aaggtctcta agcctcgctg gaaaatcatc
     2941 gacgaaaatg ggaacgagat tattttaacg aacgaccact ccgttatgat tgagcgcgat
     3001 ggtaaactta ttgaggcgaa gccgagcgaa atcaacccgg acacggatat tctgattact
     3061 attggtgagg gatcccatca tcatcaccat cattaagctt ggcactggcc gtcgttttac
     3121 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3181 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3241 gcagcctgaa tggcgaatgg cagcttggct gttttggcgg atgagataag attttcagcc
     3301 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     3361 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     3421 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     3481 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     3541 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     3601 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     3661 acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa
     3721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3781 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3841 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3901 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3961 ccccgaagaa cgtttcccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4021 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4081 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4141 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4201 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4261 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4321 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4381 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4441 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4501 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4561 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4621 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4681 tgatttaccc cggttgataa tcagaaaagc cccaaaaaca ggaagattgt ataagcaaat
     4741 atttaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4801 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc
     4861 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4921 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4981 cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5041 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5101 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5161 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt aaaaggatct aggtgaagat
     5221 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5281 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5341 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5401 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5461 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5521 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5581 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5641 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5701 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5761 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5821 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5881 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5941 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6001 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6061 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     6121 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6181 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     6241 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6301 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6361 aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt
     6421 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     6481 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     6541 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     6601 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     6661 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     6721 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     6781 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     6841 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     6901 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     6961 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgaaat t
//
