LOCUS       sequence-258899-        6356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses OT-I TCRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6356)
  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 6356)
  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-TCRB
FEATURES             Location/Qualifiers
     source          1..6356
                     /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(1947..1964)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1953..2177
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2223..2651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2310..2329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2520..2541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2660..2680)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2665..2994
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2845..2980
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2907..2926
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3061..3855
                     /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(3115..3134)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3725..3744
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4029..4150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4066..4085)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4120..4139
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4199..4215)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4199..4215)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4212..4234)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4223..4239
                     /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(4247..4253,4254..4271,4272..4277))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4292..4313
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4430..4447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4601..5189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4681..4700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5360..6151,6152..6220))
                     /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     5983..6002
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6221..6325)
                     /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 atccatgtct aacactgtcc tcgctgattc
      961 tgcctggggc atcaccctgc tatcttgggt tactgtcttt ctcttgggaa caagttcagc
     1021 agattctggg gttgtccagt ctccaagaca cataatcaaa gaaaagggag gaaggtccgt
     1081 tctgacgtgt attcccatct ctggacatag caatgtggtc tggtaccagc agactctggg
     1141 gaaggaatta aagttcctta ttcagcatta tgaaaaggtg gagagagaca aaggattcct
     1201 acccagcaga ttctcagtcc aacagtttga tgactatcac tctgaaatga acatgagtgc
     1261 cttggaactg gaggactctg ctatgtactt ctgtgccagc tctcgggcca attatgaaca
     1321 gtacttcggt cccggcacca ggctcacggt tttagaggat ctgagaaatg tgactccacc
     1381 caaggtctcc ttgtttgagc catcaaaagc agagattgca aacaaacaaa aggctaccct
     1441 cgtgtgcttg gccaggggct tcttccctga ccacgtggag ctgagctggt gggtgaatgg
     1501 caaggaggtc cacagtgggg tcagcacgga ccctcaggcc tacaaggaga gcaattatag
     1561 ctactgcctg agcagccgcc tgagggtctc tgctaccttc tggcacaatc ctcgaaacca
     1621 cttccgctgc caagtgcagt tccatgggct ttcagaggag gacaagtggc cagagggctc
     1681 acccaaacct gtcacacaga acatcagtgc agaggcctgg ggccgagcag actgtggaat
     1741 cacttcagca tcctatcatc agggggttct gtctgcaacc atcctctatg agatcctact
     1801 ggggaaggcc accctatatg ctgtgctggt cagtggcctg gtgctgatgg ccatggtcaa
     1861 gaaaaaaaat tcctaagaat tctgcagata tccagcacag tggcggccgc tcgagtctag
     1921 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1981 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2041 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2101 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2161 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2221 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2281 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2341 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2401 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2461 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2521 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2581 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2641 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2701 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2761 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2821 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2881 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2941 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3001 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3061 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3121 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3181 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3241 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3301 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3361 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3421 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3481 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3541 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3601 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3661 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3721 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3781 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3841 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3901 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3961 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4021 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4081 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4141 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4201 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4261 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4321 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4381 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4441 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4501 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4561 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4621 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4681 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4741 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4801 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4861 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4921 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4981 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5041 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5101 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5161 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5221 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5281 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5341 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5401 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5461 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5521 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5581 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5641 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5701 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5761 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5821 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5881 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5941 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6001 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6061 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6121 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6181 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6241 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6301 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
