LOCUS       sequence-274051-        6285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for the beta subunit of the mouse TCR #33 that is 
            reactive against MC38.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6285)
  AUTHORS   Gottschalk E, Aksoy BA, Aksoy P, Swiderska-syn M, Mart C, Macpherson
            L, Romeo M, Smith A, Knochelmann H, Paulos C, Timmers C, Wrangle J, 
            Rubinstein MP, Hammerbacher J
  TITLE     Discovering tumor-reactive T-cell receptors through single-cell 
            sequencing of tumor-infiltrating lymphocytes
  JOURNAL   bioRxiv 2021.11.30.470597
REFERENCE   2  (bases 1 to 6285)
  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: "bioRxiv 
            2021.11.30.470597"
COMMENT     Sequence Label: pcDNA3.1(+)-MC38-TCRB-33-MC_hammer
FEATURES             Location/Qualifiers
     source          1..6285
                     /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"
     primer_bind     complement(1876..1893)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1882..2106
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2152..2580
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2239..2258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2449..2470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2589..2609)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2594..2923
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2774..2909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2836..2855
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2990..3784
                     /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(3044..3063)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3654..3673
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3958..4079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3995..4014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4049..4068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4128..4144)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4128..4144)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4141..4163)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4152..4168
                     /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(4176..4182,4183..4200,4201..4206))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4221..4242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4359..4376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4530..5118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4610..4629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5289..6080,6081..6149))
                     /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     5912..5931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6150..6254)
                     /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 gtttaaactt aagatgggct ccattttcct cagttgcctg gccgtttgtc tcctggtggc
      961 aggtccagtc gacccgaaaa ttatccagaa accaaaatat ctggtggcag tcacagggag
     1021 cgaaaaaatc ctgatatgcg aacagtatct aggccacaat gctatgtatt ggtatagaca
     1081 aagtgctaag aagcctctag agttcatgtt ttcctacagc tatcaaaaac ttatggacaa
     1141 tcagactgcc tcaagtcgct tccaacctca aagttcaaag aaaaaccatt tagaccttca
     1201 gatcacagct ctaaagcctg atgactcggc cacatacttc tgtgccagca gccaagaggg
     1261 ggacaaccag gctccgcttt ttggagaggg gactcgactc tctgttctag aggatctgag
     1321 aaatgtgact ccacccaagg tctccttgtt tgagccatca aaagcagaga ttgcaaacaa
     1381 acaaaaggct accctcgtgt gcttggccag gggcttcttc cctgaccacg tggagctgag
     1441 ctggtgggtg aatggcaagg aggtccacag tggggtcagc acggaccctc aggcctacaa
     1501 ggagagcaat tatagctact gcctgagcag ccgcctgagg gtctctgcta ccttctggca
     1561 caatcctcga aaccacttcc gctgccaagt gcagttccat gggctttcag aggaggacaa
     1621 gtggccagag ggctcaccca aacctgtcac acagaacatc agtgcagagg cctggggccg
     1681 agcagactgt ggaatcactt cagcatccta tcatcagggg gttctgtctg caaccatcct
     1741 ctatgagatc ctactgggga aggccaccct atatgctgtg ctggtcagtg gcctggtgct
     1801 gatggccatg gtcaagaaaa aaaattccta agcggccgct cgagtctaga gggcccgttt
     1861 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1921 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1981 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2041 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2101 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2161 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2221 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2281 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2341 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2401 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2461 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2521 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2581 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2641 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2761 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2821 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     2881 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     2941 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3001 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3061 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3121 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     3181 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3241 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3301 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3361 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3421 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3481 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     3541 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3601 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3661 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3721 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     3781 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     3841 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3901 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3961 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4021 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4081 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4141 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4201 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4261 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4321 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4381 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4441 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4501 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4561 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4621 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4681 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4741 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4801 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4861 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4921 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4981 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5041 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5101 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5161 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5221 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5281 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5341 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5401 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5461 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5521 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5581 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5641 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5701 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5761 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5821 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5881 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5941 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6001 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6061 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6121 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6181 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6241 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
