LOCUS       sequence-11494-e        7584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7584)
  TITLE     Tono lab plasmid
REFERENCE   2  (bases 1 to 7584)
  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"
COMMENT     Sequence Label: pTitin
FEATURES             Location/Qualifiers
     source          1..7584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(132..200,201..992)
                     /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     complement(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2109..2126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2243..2264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2279..2284,2285..2302,2303..2309)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2317..2333
                     /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)."
     primer_bind     2322..2344
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2341..2357
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2341..2357
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2376..2396
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2378..2396
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2424..2650
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2651..2831
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2878..3003
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3496..3729
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3914..3958
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    4225..4342
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4388..4898
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4817..4835
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      4914..4923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4920..4922,4923..4925,4926..5639)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4920..4922,4923..4925,4926..5636)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4965..4986)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5226..5245)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5573..5594
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5655..6243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5708..5728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6126..6137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6330..6563
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6635..6756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6672..6691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6726..6745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6775..6910
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6942..6961)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6943..6961)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6971..6988)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6971..6987)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6980..7002)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7132..7560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7219..7238)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7429..7450
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
       61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
      541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
      601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
      661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
      721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
      781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
      841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
      901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
      961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     1801 ttgctcacat gagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg
     1861 aattcggctt tgcagtcatg ctgctagcgc tgctgctgct gaagctgctg aaggatgcct
     1921 cctgcttaga ggcagacatt tggactgact gagacgagac gcttccgtgc aagctcacgt
     1981 cactgctcgt tttaagccga attctgcaga tcatgttctt tcctgcgtta tcccctgatt
     2041 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2101 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2161 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2221 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     2281 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     2341 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     2401 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
     2461 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
     2521 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
     2581 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
     2641 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     2701 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     2761 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     2821 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     2881 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     2941 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     3001 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     3061 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     3121 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     3181 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     3241 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     3301 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     3361 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     3421 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     3481 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     3541 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     3601 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     3661 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     3721 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     3781 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     3841 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     3901 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     3961 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     4021 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     4081 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc
     4141 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa
     4201 cggatctcga cggtatcggt taacttttaa aagaaaaggg gggattgggg ggtacagtgc
     4261 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     4321 aattacaaaa attcaaaatt ttatcgatca cgagactagc ctcgagaagc ttgatatcga
     4381 attccacggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     4441 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     4501 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     4561 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     4621 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
     4681 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     4741 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     4801 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     4861 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg gtcgccacca
     4921 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4981 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5041 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     5101 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     5161 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     5221 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     5281 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     5341 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     5401 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     5461 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     5521 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     5581 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     5641 gcggccgcgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5701 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5761 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     5821 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     5881 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     5941 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     6001 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     6061 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     6121 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     6181 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     6241 cgcctggaat tcgagctcgg tacctttaag accaatgact tacaaggcag ctgtagatct
     6301 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     6361 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6421 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6481 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6541 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     6601 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     6661 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6721 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     6781 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     6841 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     6901 tttggaggcc taggcttttg cgtcgagacg tacccaattc gccctatagt gagtcgtatt
     6961 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     7021 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     7081 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc gacgcgccct
     7141 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     7201 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     7261 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     7321 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     7381 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     7441 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     7501 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     7561 ttaacaaaat attaacgttt acaa
//
