LOCUS       sequence-491288-        6237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neo-2/15-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 6237)
  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 6237)
  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: Neo-2/15-TGFa-SPMn-SpyTag003
FEATURES             Location/Qualifiers
     source          1..6237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     609..628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(847..951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1026..1045)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1217..1596
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1597..1800
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1751..1771
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1845..1864
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1845..1863
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1914..1923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2538..2585
                     /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(2810..2827)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2816..3040
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3086..3514
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3173..3192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3383..3404
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3523..3543)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3528..3857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3708..3843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3770..3789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3924..4718
                     /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(3978..3997)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4588..4607
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4892..5013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4929..4948)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4983..5002
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5062..5078)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5062..5078)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5075..5097)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5086..5102
                     /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(5110..5116,5117..5134,5135..5140))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5155..5176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5293..5310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5464..6052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5544..5563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6223..6237,1..777,778..846))
                     /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 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
       61 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
      121 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
      181 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
      241 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
      301 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
      361 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
      421 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
      481 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
      541 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
      601 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
      661 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
      721 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
      781 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
      841 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
      901 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
      961 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     1021 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     1081 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     1141 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     1201 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     1261 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1321 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1381 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1441 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1501 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1561 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1621 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     1681 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     1741 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     1801 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
     1861 agggagaccc aagctggcta gcgtttaaac gggccctcta gactcgagcg gccgccacca
     1921 tggacgctat gaagcgaggt ctgtgttgcg tgctgctgct ttgtggtgcg gtattcgtat
     1981 ccccatctta ccagggctct ggcccgaaaa aaaaaataca gctgcacgcg gaacatgctt
     2041 tgtatgatgc gttgatgata ctgaacattg ttaagacgaa tagcccgccg gctgaagaga
     2101 agctggagga ttatgccttc aactttgaac ttattcttga agagatcgca cgcctcttcg
     2161 aatctggtga ccagaaggat gaggccgaga aggcaaaacg gatgaaagag tggatgaagc
     2221 gaatcaaaac tactgcctct gaagacgagc aggaggaaat ggcgaatgct atcattacaa
     2281 tcctgcagtc ctggattttt tccggcagca gcgtcgtctc ccattttaac gattgccccg
     2341 acagccacac acaattctgt tttcatggga cctgtcggtt cttggtacag gaggataaac
     2401 ctgcttgcgt gtgccatagc ggctacgtcg gggcaagatg cgagcacgcg gatctgttgg
     2461 ctggcagcta tgacccgctc gccttggatc tcgatggtga tggtatagag acagtagccg
     2521 ctaaaggggg gagcgggcgg ggcgtacctc acattgtgat ggtcgacgcc tacaaacggt
     2581 acaagtagta gaccggtaag ctgagcagcg gctacaagga cgtgatcctg tggttcagct
     2641 tcggcgccag ctgcttcatc ctgctggcca tcgtgatggg cctggtgttc atctgcgtga
     2701 agaacggcaa catgcggtgc accatctgca tctgatgata tcgaattcca ccacactgga
     2761 ctagtggatc cgagctcggt accaagctta agtttaaacc gctgatcagc ctcgactgtg
     2821 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2881 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2941 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3001 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     3061 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3121 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3181 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3241 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3301 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3361 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3421 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3481 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3541 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3601 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3661 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3721 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3781 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3841 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3901 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3961 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4021 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4081 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4141 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4201 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4261 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4321 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4381 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4441 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4501 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4561 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4621 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4681 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4741 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4801 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4861 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4921 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4981 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5041 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5101 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5161 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5221 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5281 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5341 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5401 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5461 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5521 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5581 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5641 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5701 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5761 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5821 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5881 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5941 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6001 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6061 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6121 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6181 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaat
//
