LOCUS       sequence-403308-        4937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning single chain variable fragments displayed 
            on the human CD8a hinge and transmembrane domain (CAG promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4937)
  AUTHORS   Hamilton JR, Chen E, Perez BS, Sandoval Espinoza CR, Kang MH, 
            Trinidad M, Ngo W, Doudna JA
  TITLE     In vivo human T cell engineering with enveloped delivery vehicles.
  JOURNAL   Nat Biotechnol. 2024 Jan 11. doi: 10.1038/s41587-023-02085-z.
  PUBMED    38212493
REFERENCE   2  (bases 1 to 4937)
  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: "Nat 
            Biotechnol. 2024 Jan 11. doi: 10.1038/s41587-023-02085-z."
COMMENT     Sequence Label: pJRH-1362 scFv entry plasmid
FEATURES             Location/Qualifiers
     source          1..4937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..641
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          642..1658
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1582..1604
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1666..1685
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1713..1722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1959..1978)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2024..2079
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2078..2097)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2440..2456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2440..2456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2453..2475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2464..2480
                     /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(2488..2494,2495..2512,2513..2518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2533..2554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2613..2808
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2659..2794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2814..2948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2851..2870)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2905..2924
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3016..3033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3187..3775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3267..3286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3946..4737,4738..4806))
                     /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     4569..4588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4807..4911)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg ggggrgmsrg gcgaggggcg gggcggggcg aggcggagag
      541 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      601 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      661 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      721 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      781 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      841 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      901 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      961 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
     1021 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1081 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1141 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1201 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1261 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1321 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1381 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1441 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1501 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1561 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1621 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1681 ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat ggccttaacc gtctcctcaa
     1741 ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt
     1801 ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg
     1861 acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg gttcttctcc
     1921 tgtcactggt tatcaccctt tactgctaag ctagcagatc tttttccctc tgccaaaaat
     1981 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     2041 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     2101 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     2161 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     2221 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     2281 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     2341 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg
     2401 agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2461 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2521 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2581 ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc
     2641 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2701 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2761 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt
     2821 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2881 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     2941 tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3001 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3061 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3121 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3181 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3241 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3301 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3361 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3421 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3481 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3541 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3601 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3661 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3721 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3781 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3841 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3901 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3961 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4021 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4081 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4141 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4201 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4261 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4321 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4381 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4441 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4501 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4561 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4621 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4681 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4741 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4801 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4861 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4921 ccgaaaagtg ccacctg
//
