LOCUS       sequence-433370-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anti-HER2 H2-18 Heavy chain-SpyTag003 in mammalian cells. 
            To be paired with H2-18 light chain to form the anti-HER2 H2-18 Fab.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  AUTHORS   Driscoll CL, Keeble AH, Howarth MR
  TITLE     SpyMask enables combinatorial assembly of bispecific binders.
  JOURNAL   Nat Commun. 2024 Mar 16;15(1):2403. doi: 10.1038/s41467-024-46599-9.
  PUBMED    38493197
REFERENCE   2  (bases 1 to 6392)
  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: "Nat 
            Commun."; date: "2024-03-16"; volume: "15(1):2403. doi"; pages: " 
            10.1038/s41467-024-46599-9"
COMMENT     Sequence Label: pcDNA3.1-H2-18 Heavy chain-SpyTag003
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      931..940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1690..1737
                     /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"
     CDS             1741..1758
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1983..2000)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1989..2213
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2259..2687
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2346..2365)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2556..2577
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2696..2716)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2701..3030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2881..3016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2943..2962
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3097..3891
                     /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
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLGEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3151..3170)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3761..3780
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4065..4186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4102..4121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4156..4175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4235..4251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4235..4251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4248..4270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4259..4275
                     /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(4283..4289,4290..4307,4308..4313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4328..4349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4466..4483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4637..5225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4717..4736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5396..6187,6188..6256))
                     /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     6019..6038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6257..6361)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagcggc gccaccatgg ggatccttcc cagccctggg
      961 atgcctgcgc tgctctccct cgtgagcctt ctctccgtgc tgctgatggg ttgcgtggca
     1021 gaggtccagc ttgtccaatc aggggccgaa gtgaagaaac ccggcgagag cttaaaaatt
     1081 tcctgcaagg ggagcggtta ctcttttacg tcttactgga ttggatgggt acgtcaggct
     1141 ccgggccaag gtttagagtg gatgggatgg atttctgcgt ataatggaaa caccaattat
     1201 gcgcaaaagc tgcagggtcg cgtaaccatg accacggata cctcgacatc aaccgcctac
     1261 atggaactgc gctccctgcg ctcagatgat accgcagtat attactgcgc ccgtgagggc
     1321 gatggagctt tcgattattg ggggcagggc acgcttgtca cggtcagttc agcctccacc
     1381 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg
     1441 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
     1501 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac
     1561 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
     1621 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc taagtcttgc
     1681 gggtccgggc gtggcgtgcc tcacatcgtg atggtggacg cctacaagcg ttacaagggg
     1741 catcaccacc atcatcattg ataaaccggt aagctgagca gcggctacaa ggacgtgatc
     1801 ctgtggttca gcttcggcgc cagctgcttc atcctgctgg ccatcgtgat gggcctggtg
     1861 ttcatctgcg tgaagaacgg caacatgcgg tgcaccatct gcatctgatg atatcgaatt
     1921 ccaccacact ggactagtgg atccgagctc ggtaccaagc ttaagtttaa accgctgatc
     1981 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2041 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2101 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2161 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2221 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2281 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2341 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2401 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2461 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2521 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2581 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2641 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2701 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2761 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2821 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2881 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2941 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3001 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3061 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3121 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3181 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3241 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3301 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3361 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3421 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3481 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3541 gaagccggtc ttgtcgatca ggatgatctg ggcgaagagc atcaggggct cgcgccagcc
     3601 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3661 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3721 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3781 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3841 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3901 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3961 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4021 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4081 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4141 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4201 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4261 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4321 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4441 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4501 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4561 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4621 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4681 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4741 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4801 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4861 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4921 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4981 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5041 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5101 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5161 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5221 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5281 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5341 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5401 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5461 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5521 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5581 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5641 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5701 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5761 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5821 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5881 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5941 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6001 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6061 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6121 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6181 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6241 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6301 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6361 gcacatttcc ccgaaaagtg ccacctgacg tc
//
