LOCUS       sequence-433208-        6402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anti-HER2 Tras Heavy chain-SpyTag003 in mammalian cells. 
            To be paired with Tras light chain to form the anti-HER2 Tras NoLink
            Fab.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6402)
  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 6402)
  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-Tras heavy chain-SpyTag003
FEATURES             Location/Qualifiers
     source          1..6402
                     /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      932..941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1700..1747
                     /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             1751..1768
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1993..2010)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1999..2223
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2269..2697
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2356..2375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2566..2587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2706..2726)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2711..3040
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2891..3026
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2953..2972
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3107..3901
                     /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(3161..3180)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3771..3790
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4075..4196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4112..4131)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4166..4185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4245..4261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4245..4261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4258..4280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4269..4285
                     /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(4293..4299,4300..4317,4318..4323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4338..4359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4476..4493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4647..5235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4727..4746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5406..6197,6198..6266))
                     /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     6029..6048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6267..6371)
                     /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 cgccaccatg gggatccttc ccagccctgg
      961 gatgcctgcg ctgctctccc tcgtgagcct tctctccgtg ctgctgatgg gttgcgtagc
     1021 tggtaccgga gaggtgcagc tggtagagtc cggaggcggc ctcgtgcagc ccggtggcag
     1081 ccttaggctg agctgtgccg cgagtggctt caacatcaaa gacacctaca tccactgggt
     1141 ccgacaagcc cctgggaagg gcctggagtg ggtcgcccga atctacccca ccaacggcta
     1201 cacaaggtac gctgatagcg tgaaggggag gtttaccatt agcgcagaca ccagcaaaaa
     1261 caccgcatac ctccaaatga acagcttgcg agcggaggac accgccgtgt actactgcag
     1321 ccgatggggc ggatggggct tctatgccat ggactactgg ggccaaggca cacttgttac
     1381 cgtgtccagc gccagcacca agggcccctc cgtgttcccc ctggctccca gcagcaagag
     1441 cacctctggt ggaactgctg cgctcggttg cctggtgaag gattactttc ctgagcccgt
     1501 gacggtgagc tggaactccg gtgcgctgac cagcggagtg cacacctttc ccgccgtcct
     1561 gcaaagcagc ggcttgtact cactgagctc cgtggtgacc gtgccaagca gctctctggg
     1621 cacccagaca tacatctgca acgtgaacca taagccgagc aataccaagg tggacaagaa
     1681 ggtagagccc aagtcttgcc gtggcgtgcc tcacatcgtg atggtggacg cctacaagcg
     1741 ttacaagggg catcaccacc atcatcattg ataaaccggt aagctgagca gcggctacaa
     1801 ggacgtgatc ctgtggttca gcttcggcgc cagctgcttc atcctgctgg ccatcgtgat
     1861 gggcctggtg ttcatctgcg tgaagaacgg caacatgcgg tgcaccatct gcatctgatg
     1921 atatcgaatt ccaccacact ggactagtgg atccgagctc ggtaccaagc ttaagtttaa
     1981 accgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2041 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2101 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2161 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2221 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2281 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2341 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2401 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2461 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2521 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2581 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2641 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2701 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2761 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2821 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2881 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2941 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3001 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3061 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3121 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3181 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3241 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3301 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3361 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3421 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3481 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3541 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3601 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3661 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3721 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3781 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3841 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3901 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     3961 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4021 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4081 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4141 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4201 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4261 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4321 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4381 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4441 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
