LOCUS       sequence-258901-        7238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses OT-I TCRA linked to TCRB with the P2A self-cleaving 
            peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7238)
  AUTHORS   Aksoy P, Aksoy Bulent Arman, Czech E, Hammerbacher J
  TITLE     Viable and efficient electroporation-based genetic manipulation of 
            unstimulated human T cells
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 7238)
  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"
COMMENT     Sequence Label: pcDNA3.1(+)-OTI-TCRA_p2_TCRB
FEATURES             Location/Qualifiers
     source          1..7238
                     /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"
     CDS             1760..1816
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2829..2846)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2835..3059
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3105..3533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3192..3211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3402..3423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3542..3562)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3547..3876
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3727..3862
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3789..3808
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3943..4737
                     /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(3997..4016)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4607..4626
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4911..5032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4948..4967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5002..5021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5081..5097)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5081..5097)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5094..5116)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5105..5121
                     /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(5129..5135,5136..5153,5154..5159))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5174..5195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5312..5329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5483..6071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5563..5582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6242..7033,7034..7102))
                     /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     6865..6884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7103..7207)
                     /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 aagcttggta ccgagctcgg atccatggac aagatcctga cagcatcgtt
      961 tttactccta ggccttcacc tagctggggt gaatggccag cagcaggaga aacgtgacca
     1021 gcagcaggtg agacaaagtc cccaatctct gacagtctgg gaaggagaga ccgcaattct
     1081 gaactgcagt tatgaggaca gcacttttaa ctacttccca tggtaccagc agttccctgg
     1141 ggaaggccct gcactcctga tatccatacg ttcagtgtcc gataaaaagg aagatggacg
     1201 attcacaatc ttcttcaata aaagggagaa aaagctctcc ttgcacatca cagactctca
     1261 gcctggagac tcagctacct acttctgtgc agcaagtgac aactatcagt tgatctgggg
     1321 ctctgggacc aagctaatta taaagccaga catccagaac ccagaacctg ctgtgtacca
     1381 gttaaaagat cctcggtctc aggacagcac cctctgcctg ttcaccgact ttgactccca
     1441 aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc actgacaaaa ctgtgctgga
     1501 catgaaagct atggattcca agagcaatgg ggccattgcc tggagcaacc agacaagctt
     1561 cacctgccaa gatatcttca aagagaccaa cgccacctac cccagttcag acgttccctg
     1621 tgatgccacg ttgactgaga aaagctttga aacagatatg aacctaaact ttcaaaacct
     1681 gtcagttatg ggactccgaa tcctcctgct gaaagtagcc ggatttaacc tgctcatgac
     1741 gctgaggctg tggtcctccg ccacgaactt ctctctgtta aagcaagcag gagacgtgga
     1801 agaaaacccc ggtcctatgt ctaacactgt cctcgctgat tctgcctggg gcatcaccct
     1861 gctatcttgg gttactgtct ttctcttggg aacaagttca gcagattctg gggttgtcca
     1921 gtctccaaga cacataatca aagaaaaggg aggaaggtcc gttctgacgt gtattcccat
     1981 ctctggacat agcaatgtgg tctggtacca gcagactctg gggaaggaat taaagttcct
     2041 tattcagcat tatgaaaagg tggagagaga caaaggattc ctacccagca gattctcagt
     2101 ccaacagttt gatgactatc actctgaaat gaacatgagt gccttggaac tggaggactc
     2161 tgctatgtac ttctgtgcca gctctcgggc caattatgaa cagtacttcg gtcccggcac
     2221 caggctcacg gttttagagg atctgagaaa tgtgactcca cccaaggtct ccttgtttga
     2281 gccatcaaaa gcagagattg caaacaaaca aaaggctacc ctcgtgtgct tggccagggg
     2341 cttcttccct gaccacgtgg agctgagctg gtgggtgaat ggcaaggagg tccacagtgg
     2401 ggtcagcacg gaccctcagg cctacaagga gagcaattat agctactgcc tgagcagccg
     2461 cctgagggtc tctgctacct tctggcacaa tcctcgaaac cacttccgct gccaagtgca
     2521 gttccatggg ctttcagagg aggacaagtg gccagagggc tcacccaaac ctgtcacaca
     2581 gaacatcagt gcagaggcct ggggccgagc agactgtgga atcacttcag catcctatca
     2641 tcagggggtt ctgtctgcaa ccatcctcta tgagatccta ctggggaagg ccaccctata
     2701 tgctgtgctg gtcagtggcc tggtgctgat ggccatggtc aagaaaaaaa attcctaaga
     2761 attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg
     2821 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2881 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2941 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3001 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3061 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3541 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3601 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3661 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3721 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3781 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3841 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3901 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3961 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4021 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4081 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     4141 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4201 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4261 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4321 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4381 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4441 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4501 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4561 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4621 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4681 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4741 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4801 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4861 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4921 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4981 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5041 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5101 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5161 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5221 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5281 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5401 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5461 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5521 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5581 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5641 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5701 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5761 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5821 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5881 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5941 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6001 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6061 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6121 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6181 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6241 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6301 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6361 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6421 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6481 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6541 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6601 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6661 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6721 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6781 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6841 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6901 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6961 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7021 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7081 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7141 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7201 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
