LOCUS       sequence-443923-        5086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression GST fusion CD28 CAR hinge (I114-P152) in Escherichia 
            coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5086)
  AUTHORS   Folimonova V, Chen X, Negi H, Schwieters CD, Li J, Byrd RA, Taylor 
            N, Youkharibache P, Walters KJ
  TITLE     CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits 
            local structure and conformational exchange amidst global disorder.
  JOURNAL   Commun Biol. 2024 Aug 31;7(1):1072. doi: 10.1038/s42003-024-06770-w.
  PUBMED    39217198
REFERENCE   2  (bases 1 to 5086)
  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: "Commun 
            Biol."; date: "2024-08-31"; volume: "7(1):1072. doi"; pages: " 
            10.1038/s42003-024-06770-w"
COMMENT     Sequence Label: pGEX6P1-CD28 CAR hinge (I114 – P152)
FEATURES             Location/Qualifiers
     source          1..5086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     419..438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(657..761)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     829..847
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     992..1014
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1209..1232)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1239..1892)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1259..1281)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    1915..1931
                     /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(1939..1945,1946..1961,1962..1967))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(2069..2596)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     2545..2567
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2559..2576
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2560..2576
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2592..2608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2592..2608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2616..2632
                     /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(2640..2646,2647..2664,2665..2670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2719..3801)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3763..3782
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3802..3879)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(3952..3969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4123..4711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4203..4222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4882..5086,1..587,588..656))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
       61 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
      121 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      181 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      241 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      301 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      361 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
      421 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      481 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      541 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      601 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      661 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      721 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      781 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
      841 acgaggccct ttcgtcttca agaattatac actccgctat cgctacgtga ctgggtcatg
      901 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
      961 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     1021 ccgtcatcac cgaaacgcgc gaggcagatc gtcagtcagt cacgatgcgg ccgctcgagt
     1081 tacggtttgc ttggacccgg gaacaacggg gacgggcaca ggtgcttacc cttaacatgg
     1141 ataatggtgc cattgctttt ctcgttgtct agataaggtg gggggtacat tacttctatg
     1201 gatcccaggg gcccctggaa cagaacttcc agatccgatt ttggaggatg gtcgccacca
     1261 ccaaacgtgg cttgccagcc ctgcaaaggc catgctatat acttgctgga tttcaagtac
     1321 ttatcaattt gtgggatagc ttcaatacgt tttttaaaac aaactaattt tgggaacgca
     1381 tccaggcaca ttgggtccat gtataaaaca acatcaagag cgtcatacaa catgaagtca
     1441 ggatgggtta catgatcacc atttaaatat gttttatgac ataaacgatc ttcgaacatt
     1501 ttcagcattt caggtagctt gctaagaaaa tcaactttga gagtttcaaa gtctttacta
     1561 tatgcaattc tcgaaacacc gtatctaata tccaaaaccg ctccttcaag cattgaaatc
     1621 tctgcacgct cttttggaca accacccaac atgttgtgct tgtcagctat ataacgtatg
     1681 atggccatag actgtgttaa tttaacatca ccatcaatat aataaggaag attgggaaac
     1741 tccaaaccca attcaaactt tttgtttcgc catttatcac cttcatcgcg ctcatacaaa
     1801 tgctcttcat atttttcttc aagatattcc aaaagaagtc gagtgggttg cacaaggccc
     1861 ttaattttcc aataacctag tataggggac atgaatactg tttcctgtgt gaaattgtta
     1921 tccgctcaca attccacaca ttatacgagc cgatgattaa ttgtcaacag ctcatttcag
     1981 aatatttgcc agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa cgggagtgcg
     2041 ccttgagcga cacgaattat gcagtgattt acgacctgca cagccatacc acagcttccg
     2101 atggctgcct gacgccagaa gcattggtgc accgtgcagt cgataagcta attccaacgc
     2161 catcaaaaat aattcgcgtc tggccttcct gtagccagct ttcatcaaca ttaaatgtga
     2221 gcgagtaaca acccgtcgga ttctccgtgg gaacaaacgg cggattgacc gtaatgggat
     2281 aggtcacgtt ggtgtagatg ggcgcatcgt aaccgtgcat ctgccagttt gaggggacga
     2341 cgacagtatc ggcctcagga agatcgcact ccagccagct ttccggcacc gcttctggtg
     2401 ccggaaacca ggcaaagcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2461 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2521 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     2581 ccgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2641 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactt
     2701 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2761 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctagcgtggt
     2821 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     2881 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     2941 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     3001 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     3061 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     3121 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     3181 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     3241 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     3301 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     3361 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     3421 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     3481 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     3541 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     3601 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     3661 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     3721 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     3781 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     3841 tgccataccg cgaaaggttt tgcgccattc gatggtgtcg gaatttatgc ggtgtgaaat
     3901 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     3961 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4021 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4081 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4141 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4201 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4261 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4321 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4381 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4441 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4501 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4561 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4621 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4681 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4741 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4801 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4861 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4921 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4981 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5041 tccagattta tcagcaataa accagccagc cggaagggcc gagcgc
//
