LOCUS       sequence-242292-        6393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian codon optimized N-intein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6393)
  AUTHORS   Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW
  TITLE     Design of a Split Intein with Exceptional Protein Splicing Activity.
  JOURNAL   J Am Chem Soc. 2016 Feb 24;138(7):2162-5. doi: 10.1021/jacs.5b13528.
            Epub 2016 Feb 8.
  PUBMED    26854538
REFERENCE   2  (bases 1 to 6393)
  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: 
            "10.1021/jacs.5b13528"; journalName: "J Am Chem Soc"; date: 
            "2016-02-24- 24"; volume: "138"; issue: "7"; pages: "2162-5"
COMMENT     Sequence Label: HC-CfaN
FEATURES             Location/Qualifiers
     source          1..6393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    288..309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(324..329,330..347,348..354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    362..378
                     /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     367..389
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     386..402
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     386..402
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        431..810
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        811..1014
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     965..985
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1011..1035
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1245..1263
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1245..1262
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1276..1295
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             3065..3082
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      3110..3119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3123..3257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3214..3233
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3326..3683
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3326..3517
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3349..3369)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3534..3669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3703..3720)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3703..3719)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3712..3734)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3932..4387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4019..4038)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4229..4250
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4404..4423)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4523..4545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4583..4601)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4669..4773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4774..4842,4843..5634)
                     /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     complement(4992..5011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5805..6393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6294..6313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atccaactgg
      241 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      301 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      361 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaattaattc
      421 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      481 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      541 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      601 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      661 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      721 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      781 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      841 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      901 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      961 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
     1021 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
     1081 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
     1141 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
     1201 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     1261 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
     1321 tcggttctat cgattgaatt ccaccatggg atggtcatgt atcatccttt ttctagtagc
     1381 aactgcaact ggagtacatt cagaggtgaa gctgttggaa tctggaggag gtttggtaca
     1441 gccggggggt tctctgagac tctcctgtgc agcttctgga ttcaccttca atgatttcta
     1501 catgaactgg atccgccagc ctccagggca ggcacctgag tggttgggtg ttattagaaa
     1561 caaaggtaat ggttacacaa cagaggtcaa tacatctgtg aaggggcggt tcaccatctc
     1621 cagagataat acccaaaaca tcctctatct tcaaatgaac agcctgagag ctgaggacac
     1681 cgccatttac tactgtgcaa gaggcggtcc ttattactac agtggtgacg acgcccctta
     1741 ctggggccaa ggagtcatgg tcacagtctc ctcagccacc accaagggcc catctgtcta
     1801 tccactggcc cctggatctg ctgcccaaac taactccatg gtgaccctgg gatgcctggt
     1861 caagggctat ttccctgagc cagtgacagt gacctggaac tctggatccc tgtccagcgg
     1921 tgtgcacacc ttcccagctg tcctgcagtc tgacctctac actctgagca gctcagtgac
     1981 tgtcccctcc agcacctggc ccagcgagac cgtcacctgc aacgttgccc acccggccag
     2041 cagcaccaag gtggacaaga aaattgtgcc cagggattgt ggttgtaagc cttgcatatg
     2101 tacagtccca gaagtatcat ctgtcttcat cttcccccca aagcccaagg atgtgctcac
     2161 cattactctg actcctaagg tcacgtgtgt tgtggtagca atcagcaagg atgatcccga
     2221 ggtccagttc agctggtttg tagatgatgt ggaggtgcac acagctcaga cgcaaccccg
     2281 ggaggagcag ttcaacagca ctttccgctc agtcagtgaa cttcccatca tgcaccagga
     2341 ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt gcagctttcc ctgcccccat
     2401 cgagaaaacc atctccaaaa ccaaaggcag accgaaggct ccacaggtgt acaccattcc
     2461 acctcccaag gagcagatgg ccaaggataa agtcagtctg acctgcatga taacagactt
     2521 cttccctgaa gacattactg tggagtggca gtggaatggg cagccagcgg agaactacaa
     2581 gaacactcag cccatcatgg acacagatgg ctcttacttc gtctacagca agctcaatgt
     2641 gcagaagagc aactgggagg caggaaatac tttcacctgc tctgtgttac atgagggcct
     2701 gcacaaccac catactgaga agagcctctc ccactctcct ggtaaagcta gcggaggctg
     2761 cctgagctac gacaccgaga tcctgaccgt ggagtacggc ttcctgccca tcggcaagat
     2821 cgtggaggag cgcatcgagt gcaccgtgta caccgtggac aagaacggct tcgtgtacac
     2881 ccagcccatc gcccagtggc acaaccgcgg cgagcaggag gtgttcgagt actgcctgga
     2941 ggacggcagc atcatccgcg ccaccaagga ccacaagttc atgaccaccg acggccagat
     3001 gctgcccatc gacgagatct tcgagcgcgg cctggacctg aagcaggtgg acggcctgcc
     3061 cgggcaccac caccaccacc acgggtaata gcggccgctc ccaagcttgg ccgccatggc
     3121 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3181 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3241 cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg
     3301 aaagaggaac ttggttaggt accttctgag gcggaaagaa ccatctgtgg aatgtgtgtc
     3361 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3421 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3481 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3541 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3601 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3661 ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca
     3721 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     3781 tttcgccagt tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     3841 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3901 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3961 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4021 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4081 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4141 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4201 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4261 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4321 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4381 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4441 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4501 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4561 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4621 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4681 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4741 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4801 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4861 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4921 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4981 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5041 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5101 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5161 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5221 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5281 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5341 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5401 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5461 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5521 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5581 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5641 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5701 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5761 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5821 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5881 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5941 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6001 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6061 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6121 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6181 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6241 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6301 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6361 tgatgctcgt caggggggcg gagcctatgg aaa
//
