LOCUS       sequence-348678-        6168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for the alpha subunit of the mouse TCR #30 that is 
            reactive against MC38.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6168)
  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 6168)
  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-TCRA-30-picky_sticky
FEATURES             Location/Qualifiers
     source          1..6168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1525..1542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1531..1755
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1801..2229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1888..1907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2098..2119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2238..2258)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2243..2572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2423..2558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2485..2504
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2639..3433
                     /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(2693..2712)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3303..3322
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3607..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3644..3663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3698..3717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3777..3793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3777..3793)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3790..3812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3801..3817
                     /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(3825..3831,3832..3849,3850..3855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3870..3891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4008..4025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4179..4767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4259..4278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4938..5729,5730..5798))
                     /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     5561..5580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5799..5903)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5978..5997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaaga tgaagaggct gctgtgttct ctgctggggc ttctgtgcac
      721 ccaggtttgc tgggtgaaag gacagcaagt gcagcagagt cccgcatcct tggttctgca
      781 ggagggggag aacgcagagc tgcagtgtaa cttttcctcc acagcaaccc ggctgcagtg
      841 gttttaccaa catcctgggg gaagactcgt cagcctgttc tacaatcctt ctgggacaaa
      901 gcacactgga agactgacat ccaccacagt cactaacgaa cgtcgcagct ctttgcacat
      961 ttcctcctcc cagacaacag actcaggcac ttatttctgt gctatagatc cttataacca
     1021 ggggaagctt atctttggac agggaaccaa gttatctatc aagcccaaca tccagaaccc
     1081 agaacctgct gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt
     1141 caccgacttt gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac
     1201 tgacaaaact gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg
     1261 gagcaaccag acaagcttca cctgccaaga tatcttcaaa gagaccaacg ccacctaccc
     1321 cagttcagac gttccctgtg atgccacgtt gactgagaaa agctttgaaa cagatatgaa
     1381 cctaaacttt caaaacctgt cagttatggg actccgaatc ctcctgctga aagtagccgg
     1441 atttaacctg ctcatgacgc tgaggctgtg gtcctcctaa gcggccgctc gagtctagag
     1501 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     1561 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     1621 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     1681 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     1741 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     1801 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1861 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1921 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1981 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2041 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2101 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2161 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2221 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2281 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2341 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2401 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2461 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2521 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2581 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     2641 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2701 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2761 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2821 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     2881 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     2941 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3001 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3061 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3121 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3181 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3241 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3301 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3361 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3421 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3481 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3541 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3601 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3661 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3721 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3781 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3841 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3901 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3961 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4141 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4801 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4861 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4921 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4981 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5041 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5101 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5161 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5221 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5281 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5341 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5401 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5461 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5521 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5581 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5641 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5701 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5761 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5821 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5881 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5941 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     6001 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     6061 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6121 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
