LOCUS       sequence-459829-        7157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for expression of GoI with Bi-cistronic expression 
            ZsGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  AUTHORS   Hanggi K, Li J, Gangadharan A, Liu X, Celias DP, Osunmakinde O, 
            Keske A, Davis J, Ahmad F, Giron A, Anadon CM, Gardner A, DeNardo 
            DG, Shaw TI, Beg AA, Yu X, Ruffell B
  TITLE     Interleukin-1alpha release during necrotic-like cell death generates
            myeloid-driven immunosuppression that restricts anti-tumor immunity.
  JOURNAL   Cancer Cell. 2024 Nov 13:S1535-6108(24)00402-1. doi: 
            10.1016/j.ccell.2024.10.014.
  PUBMED    39577420
REFERENCE   2  (bases 1 to 7157)
  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: "Cancer 
            Cell. 2024 Nov 13:S1535-6108(24)00402-1. doi: 
            10.1016/j.ccell.2024.10.014."
COMMENT     Sequence Label: pHIV-EF1a-2A-ZsGreen
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2624..3802
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2855..3793
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3750..3770
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3862..3915
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3928..4623
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     primer_bind     complement(4637..4653)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4638..4654)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5163..5343
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5405..5993)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5485..5504)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6164..6955,6956..7024))
                     /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     6787..6806
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7025..7129)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagcg tgaggctccg gtgcccgtca
     2641 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2701 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2761 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     2821 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
     2881 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
     2941 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     3001 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     3061 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     3121 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     3181 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     3241 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     3301 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     3361 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     3421 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     3481 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     3541 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     3601 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     3661 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     3721 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     3781 tcttccattt caggtgtcgt gagcggccgc tgaactgaat tcatcgacgt taactattct
     3841 agagtacccg ggctaggatc cgagggcaga ggaagtcttc taacatgcgg tgacgtggag
     3901 gagaatcccg gccctatggc cacaaccatg gcccagtcca agcacggcct gaccaaggag
     3961 atgaccatga agtaccgcat ggagggctgc gtggacggcc acaagttcgt gatcaccggc
     4021 gagggcatcg gctacccctt caagggcaag caggccatca acctgtgcgt ggtggagggc
     4081 ggccccttgc ccttcgccga ggacatcttg tccgccgcct tcatgtacgg caaccgcgtg
     4141 ttcaccgagt acccccagga catcgtcgac tacttcaaga actcctgccc cgccggctac
     4201 acctgggacc gctccttcct gttcgaggac ggcgccgtgt gcatctgcaa cgccgacatc
     4261 accgtgagcg tggaggagaa ctgcatgtac cacgagtcca agttctacgg cgtgaacttc
     4321 cccgccgacg gccccgtgat gaagaagatg accgacaact gggagccctc ctgcgagaag
     4381 atcatccccg tgcccaagca gggcatcttg aagggcgacg tgagcatgta cctgctgctg
     4441 aaggacggtg gccgcttgcg ctgccagttc gacaccgtgt acaaggccaa gtccgtgccc
     4501 cgcaagatgc ccgactggca cttcatccag cacaagctga cccgcgagga ccgcagcgac
     4561 gccaagaacc agaagtggca cctgaccgag cacgccatcg cctccggctc cgccttgccc
     4621 taaggatcgg ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa
     4681 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg
     4741 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg
     4801 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4861 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     4921 attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct
     4981 acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc
     5041 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt
     5101 ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta
     5161 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     5221 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     5281 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     5341 gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5401 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5461 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5521 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5581 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5641 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5701 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5761 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5821 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5881 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5941 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6001 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6061 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6121 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6181 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6241 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6301 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6361 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6421 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6481 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6541 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6601 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6661 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6721 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6781 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6841 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6901 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6961 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7021 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7081 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7141 gaaaagtgcc acctgac
//
