LOCUS       sequence-274030-        6294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for the beta subunit of the mouse TCR #31 that is 
            reactive against MC38.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6294)
  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 6294)
  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-31-murderous_crow
FEATURES             Location/Qualifiers
     source          1..6294
                     /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(1885..1902)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1891..2115
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2161..2589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2248..2267)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2458..2479
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2598..2618)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2603..2932
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2783..2918
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2845..2864
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2999..3793
                     /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(3053..3072)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3663..3682
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3967..4088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4004..4023)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4058..4077
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4137..4153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4137..4153)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4150..4172)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4161..4177
                     /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(4185..4191,4192..4209,4210..4215))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4230..4251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4368..4385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4539..5127)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4619..4638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5298..6089,6090..6158))
                     /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     5921..5940
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6159..6263)
                     /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 aagatgaaca agtgggtttt ctgctgggta accctttgtc tccttactgt
      961 agagaccaca catggtgatg gtggcatcat tactcagaca cccaaattcc tgattggtca
     1021 ggaagggcaa aaactgacct tgaaatgtca acagaatttc aatcatgata caatgtactg
     1081 gtaccgacag gattcaggga aaggattgag actgatctac tattcaataa ctgaaaacga
     1141 tcttcaaaaa ggcgatctat ctgaaggcta tgatgcgtct cgagagaaga agtcatcttt
     1201 ttctctcact gtgacatctg cccagaagaa cgagatggcc gtttttctct gtgccagcag
     1261 tataggactg gggggaatgg aacagtactt cggtcccggc accaggctca cggttttaga
     1321 ggatctgaga aatgtgactc cacccaaggt ctccttgttt gagccatcaa aagcagagat
     1381 tgcaaacaaa caaaaggcta ccctcgtgtg cttggccagg ggcttcttcc ctgaccacgt
     1441 ggagctgagc tggtgggtga atggcaagga ggtccacagt ggggtcagca cggaccctca
     1501 ggcctacaag gagagcaatt atagctactg cctgagcagc cgcctgaggg tctctgctac
     1561 cttctggcac aatcctcgaa accacttccg ctgccaagtg cagttccatg ggctttcaga
     1621 ggaggacaag tggccagagg gctcacccaa acctgtcaca cagaacatca gtgcagaggc
     1681 ctggggccga gcagactgtg gaatcacttc agcatcctat catcaggggg ttctgtctgc
     1741 aaccatcctc tatgagatcc tactggggaa ggccacccta tatgctgtgc tggtcagtgg
     1801 cctggtgctg atggccatgg tcaagaaaaa aaattcctaa gcggccgctc gagtctagag
     1861 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     1921 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     1981 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2041 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2101 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2161 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2221 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2281 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2341 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2401 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2461 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2521 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2581 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2641 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2701 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2761 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2821 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2881 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2941 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3001 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3061 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3121 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3181 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3241 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3301 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3361 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3421 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3481 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3541 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3601 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3661 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3721 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3781 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3841 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3901 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3961 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4021 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4081 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4141 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4201 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4261 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4321 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4381 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4441 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4501 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4561 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4621 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4681 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4741 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4801 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4861 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4921 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4981 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5041 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5101 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5161 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5221 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5281 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5341 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5401 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5461 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5521 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5581 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5641 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5701 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5761 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5821 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5881 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5941 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6001 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6061 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6121 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6181 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6241 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
