LOCUS       sequence-346253-        6321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Pmel-1 TCR beta chain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6321)
  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 6321)
  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(+)_Pmel-1_TCRB
FEATURES             Location/Qualifiers
     source          1..6321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..30
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(149..169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        154..483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      334..469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     396..415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             550..1344
                     /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(604..623)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1214..1233
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1518..1639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1555..1574)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1609..1628
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1688..1704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1688..1704)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1701..1723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1712..1728
                     /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(1736..1742,1743..1760,1761..1766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1781..1802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1919..1936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2090..2678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2170..2189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2849..3640,3641..3709))
                     /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     3472..3491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3710..3814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3889..3908)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4080..4459
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4460..4663
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4614..4634
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4708..4727
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4708..4726
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5757..5774)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5763..5987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6033..6321,1..140)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6120..6139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
       61 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
      421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
      481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
      541 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
      601 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
      661 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
      721 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
      781 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
      841 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
      901 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
      961 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     1021 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     1081 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     1141 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     1201 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     1261 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     1321 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     1381 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     1441 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     1501 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1561 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1621 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     1681 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1741 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1801 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1861 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1921 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1981 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2041 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2101 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2161 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2221 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2281 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2341 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2401 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2461 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2521 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2581 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2641 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2701 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2761 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2821 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2881 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2941 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3001 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3061 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3121 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3181 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3241 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3301 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3361 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3421 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3481 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3541 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3601 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3661 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3781 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     3841 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     3901 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     3961 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     4021 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     4081 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     4141 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     4201 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     4261 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     4321 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     4381 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     4441 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     4501 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     4561 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     4621 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     4681 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     4741 ctagcgttta aacttaagat gggcaccagg cttcttggct gggcagtggg cccgcggctt
     4801 cttggctggg cagtgttctg tctccttgac acagtactgt ctgaagctgg agtcacccag
     4861 tctcccagat atgcagtcct acaggaaggg caagctgttt ccttttggtg tgaccctatt
     4921 tctggacatg atacccttta ctggtatcag cagcccagag accaggggcc ccagcttcta
     4981 gtttactttc gggatgaggc tgttatagat aattcacagt tgccctcgga tcgattttct
     5041 gctgtgaggc ctaaaggaac taactccact ctcaagatcc agtctgcaaa gcagggcgac
     5101 acagccacct atctctgtgc cagcagtttc cacagggact ataattcgcc cctctacttt
     5161 gcggcaggca cccggctcac tgtgacagag gatctgagaa atgtgactcc acccaaggtc
     5221 tccttgtttg agccatcaaa agcagagatt gcaaacaaac aaaaggctac cctcgtgtgc
     5281 ttggccaggg gcttcttccc tgaccacgtg gagctgagct ggtgggtgaa tggcaaggag
     5341 gtccacagtg gggtcagcac ggaccctcag gcctacaagg agagcaatta tagctactgc
     5401 ctgagcagcc gcctgagggt ctctgctacc ttctggcaca atcctcgcaa ccacttccgc
     5461 tgccaagtgc agttccatgg gctttcagag gaggacaagt ggccagaggg ctcacccaaa
     5521 cctgtcacac agaacatcag tgcagaggcc tggggccgag cagactgtgg gattacctca
     5581 gcatcctatc aacaaggggt cttgtctgcc accatcctct atgagatcct gctagggaaa
     5641 gccaccctgt atgctgtgct tgtcagtaca ctggtggtga tggctatggt caaaagaaag
     5701 aattcatgat aagcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     5761 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5821 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     5881 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     5941 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     6001 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     6061 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6241 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6301 tttgacgttg gagtccacgt t
//
