LOCUS       sequence-491286-        6984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tPA-TGFa-SPMn-SpyTag003 in mammalian cells for 
            calcium-induced NeissLock protein ligation to epidermal growth 
            factor receptor (EGFR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6984)
  AUTHORS   Lim SYT, Keeble AH, Howarth MR
  TITLE     Split NeissLock with Spy-Acceleration Arms Mammalian Proteins for 
            Anhydride-Mediated Cell Ligation.
  JOURNAL   ACS Chem Biol. 2025 Sep 15. doi: 10.1021/acschembio.5c00515.
  PUBMED    40947984
REFERENCE   2  (bases 1 to 6984)
  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: "ACS Chem 
            Biol. 2025 Sep 15. doi: 10.1021/acschembio.5c00515."
COMMENT     Sequence Label: tPA-TGFa-SPMn-SpyTag003
FEATURES             Location/Qualifiers
     source          1..6984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     270..289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(508..612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(687..706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        878..1257
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1258..1461
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1412..1432
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1506..1525
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1506..1524
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1575..1584
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2946..2993
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     primer_bind     complement(3218..3235)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3224..3448
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3494..3922
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3581..3600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3791..3812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3931..3951)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3936..4265
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4116..4251
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4178..4197
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4332..5126
                     /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(4386..4405)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4996..5015
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5300..5421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5337..5356)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5391..5410
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5470..5486)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5470..5486)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5483..5505)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5494..5510
                     /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(5518..5524,5525..5542,5543..5548))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5563..5584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5701..5718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5872..6460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5952..5971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6631..6984,1..438,439..507))
                     /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"
ORIGIN
        1 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
       61 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
      121 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
      181 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
      241 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
      301 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      361 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
      421 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
      481 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
      541 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
      601 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
      661 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      721 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
      781 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
      841 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
      901 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      961 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     1021 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     1081 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     1141 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1201 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     1261 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     1321 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1381 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1441 gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc
     1501 gaaattaata cgactcacta tagggagacc caagctggct agcgtttaaa cgggccctct
     1561 agactcgagc ggccgccacc atggacgcta tgaagcgagg tctgtgttgc gtgctgctgc
     1621 tttgtggtgc ggtattcgta tccccatctt accagggcaa tagcgattgc tatttcggca
     1681 atggctctgc ctatcggggc acccattcac tgaccgagtc cggggcttct tgccttccct
     1741 ggaactccat gatactcatt gggaaagtct acacagcgca gaacccaagc gcgcaagcgc
     1801 tcggccttgg gaagcacaat tattgcagaa accccgacgg cgacgctaaa ccatggtgcc
     1861 atgtcctcaa gaatcgcagg cttacgtggg agtattgcga tgttccttct tgttccacat
     1921 gtggtttgcg ccaatacagc caaccgcaat ttaggattaa gggcggcctt ttcgccgata
     1981 tcgcatcaca cccgtggcaa gctgcaatct ttgccgctgc agccgctagt ccaggggagc
     2041 gctttctttg tggcggtatc cttatatctt cctgctggat actcagcgcc gcccattgct
     2101 tccaagaacg atttccaccg catcatctga ccgtcatact tggtaggacg tatagggtag
     2161 tccccgggga ggaagaacaa aaattcgagg tagaaaaata catcgtacat aaggagttcg
     2221 acgatgatac ttacgataac gatatagccc tgctccagct gaagagcgac agtagtagat
     2281 gtgctcagga atcctctgta gttcggaccg tctgtcttcc gccggcggac cttcaacttc
     2341 cggactggac cgagtgcgaa ttgagcggct acggtaagca cgaagcactc tccccatttt
     2401 actccgagcg gcttaaggaa gctcacgtgc gactctaccc cagctcacgg tgcacatcac
     2461 agcacttgct gaatcggact gttaccgaca atatgctctg tgcgggagat accaggtcag
     2521 gtggacctca agctaacctt cacgatgcgt gccagggtga ttcaggtggt ccgcttgtct
     2581 gtcttaacga cgggcgaatg accctggttg ggatcatatc atgggggctg gggtgtgggc
     2641 aaaaagatgt gccgggagta tataccaagg ttacgaacta cctggattgg atcagagaca
     2701 atatgcgccc cggcagcagc gtcgtctccc attttaacga ttgccccgac agccacacac
     2761 aattctgttt tcatgggacc tgtcggttct tggtacagga ggataaacct gcttgcgtgt
     2821 gccatagcgg ctacgtcggg gcaagatgcg agcacgcgga tctgttggct ggcagctatg
     2881 acccgctcgc cttggatctc gatggtgatg gtatagagac agtagccgct aaagggggga
     2941 gcgggcgggg cgtacctcac attgtgatgg tcgacgccta caaacggtac aagtagtaga
     3001 ccggtaagct gagcagcggc tacaaggacg tgatcctgtg gttcagcttc ggcgccagct
     3061 gcttcatcct gctggccatc gtgatgggcc tggtgttcat ctgcgtgaag aacggcaaca
     3121 tgcggtgcac catctgcatc tgatgatatc gaattccacc acactggact agtggatccg
     3181 agctcggtac caagcttaag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc
     3241 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3301 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3361 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3421 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3481 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3541 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3901 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3961 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4021 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4081 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4141 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4201 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4261 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4321 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4381 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4441 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4501 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4561 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4621 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4681 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4741 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4801 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4861 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4921 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4981 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5041 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5101 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5161 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5221 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5281 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5341 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5401 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5461 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5521 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5581 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5641 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5701 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5761 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5821 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5881 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5941 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6001 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6061 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6121 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6181 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6241 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6301 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6361 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6421 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6481 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6541 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6601 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6661 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6721 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6781 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6841 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6901 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6961 gtcacgctcg tcgtttggta tggc
//
