LOCUS       sequence-274047-        6282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for the beta subunit of the mouse TCR #32 that is 
            reactive against MC38.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6282)
  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 6282)
  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-32-riot_punisher
FEATURES             Location/Qualifiers
     source          1..6282
                     /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(1873..1890)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1879..2103
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2149..2577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2236..2255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2446..2467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2586..2606)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2591..2920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2771..2906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2833..2852
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2987..3781
                     /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(3041..3060)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3651..3670
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3955..4076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3992..4011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4046..4065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4125..4141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4125..4141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4138..4160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4149..4165
                     /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(4173..4179,4180..4197,4198..4203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4218..4239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4356..4373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4527..5115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4607..4626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5286..6077,6078..6146))
                     /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     5909..5928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6147..6251)
                     /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 ccaggctctt tctggtcttg agcctcctgt gtacaaaaca
      961 catggaggct gcagtcaccc aaagccctag aaacaaggtg acagtaacag gaggaaacgt
     1021 gacattgagc tgtcgccaga ctaatagcca caactacatg tactggtatc ggcaggacac
     1081 tgggcatggg ctgaggctga tccattactc atatggtgct ggcaaccttc aaataggaga
     1141 tgtccctgat gggtacaagg ccaccagaac aacgcaagaa gacttcttcc tcctgctgga
     1201 attggcttct ccctctcaga catctttgta cttctgtgcc agcagtaggg acaattccta
     1261 taattcgccc ctctactttg cggcaggcac ccggctcact gtgacagagg atctgagaaa
     1321 tgtgactcca cccaaggtct ccttgtttga gccatcaaaa gcagagattg caaacaaaca
     1381 aaaggctacc ctcgtgtgct tggccagggg cttcttccct gaccacgtgg agctgagctg
     1441 gtgggtgaat ggcaaggagg tccacagtgg ggtcagcacg gaccctcagg cctacaagga
     1501 gagcaattat agctactgcc tgagcagccg cctgagggtc tctgctacct tctggcacaa
     1561 tcctcgaaac cacttccgct gccaagtgca gttccatggg ctttcagagg aggacaagtg
     1621 gccagagggc tcacccaaac ctgtcacaca gaacatcagt gcagaggcct ggggccgagc
     1681 agactgtgga atcacttcag catcctatca tcagggggtt ctgtctgcaa ccatcctcta
     1741 tgagatccta ctggggaagg ccaccctata tgctgtgctg gtcagtggcc tggtgctgat
     1801 ggccatggtc aagaaaaaaa attcctaagc ggccgctcga gtctagaggg cccgtttaaa
     1861 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     1921 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     1981 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2041 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2101 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2161 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2221 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2281 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2341 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2401 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2461 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2521 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     2581 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2641 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2701 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2761 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2821 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     2881 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     2941 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3001 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3061 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3121 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3181 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3241 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3301 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3361 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3421 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3481 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3541 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3601 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3661 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3721 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3781 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     3841 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3901 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3961 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4021 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4081 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4141 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4201 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5161 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6181 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6241 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
