LOCUS       sequence-417795-        3744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Linker fragment for direct rescue of SARS-CoV-2, including 5' 
            Promoter and 3' Termination signals and 100bp SARS-CoV-2 homology 
            regions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3744)
  AUTHORS   Kipfer ET, Hauser D, Lett MJ, Otte F, Urda L, Zhang Y, Lang CMR, 
            Chami M, Mittelholzer C, Klimkait T
  TITLE     Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a 
            novel reverse genetics platform.
  JOURNAL   Elife. 2023 Nov 21;12:RP89035. doi: 10.7554/eLife.89035.
  PUBMED    37988285
REFERENCE   2  (bases 1 to 3744)
  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: "Elife."; 
            date: "2023-11-21"; volume: "12:RP89035. doi"; pages: " 
            10.7554/eLife.89035"
COMMENT     Sequence Label: pUC19_CoV-2_linker
FEATURES             Location/Qualifiers
     source          1..3744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    237..258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(273..278,279..296,297..303)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    311..327
                     /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     316..338
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     335..351
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     335..351
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    592..713
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(629..648)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     683..702
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        775..1154
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1155..1358
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1309..1329
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(1479..1496)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1479..1495)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1488..1510)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1704..1723)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1823..1845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1883..1901)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1969..2073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2074..2142,2143..2934)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPSGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2292..2311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3105..3693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3594..3613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
       61 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      121 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
      181 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      241 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      301 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      361 gcctgctagg gagagctgcc tatatggaag agccctaatg tgtaaaatta attttagtag
      421 tgctatcccc atgtgatttt aatagcttct taggagaatg acaaaaaaaa aaaaaaaaaa
      481 aaaaaaaaaa aaaaaaaaaa aaaaaaaggc cggcatggtc ccagcctcct cgctggcgcc
      541 ggctgggcaa cattccgagg ggaccgtccc ctcggtaatg gcgaatggga caacttgttt
      601 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
      661 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
      721 tggcggccgc tgagacgggt ctccctaacg atgtacgggc cagatatacg cgttgacatt
      781 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      841 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      901 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      961 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     1021 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     1081 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     1141 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     1201 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     1261 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     1321 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtatattag
     1381 gtttatacct tcccaggtaa caaaccaacc aactttcgat ctcttgtaga tctgttctct
     1441 aaacgaactt taaaatctgt gtggctgtca ctcggctgac tggccgtcgt tttacaacgt
     1501 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     1561 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     1621 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     1681 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1741 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1801 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1861 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1921 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1981 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2041 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2101 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2161 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2221 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2281 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2341 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2401 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2461 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2521 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2581 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2641 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2701 gaggcggata aagttgcagg accacttctg cgctcggccc ttccgtctgg ctggtttatt
     2761 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2821 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2881 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2941 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3001 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3061 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3121 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3181 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3241 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3301 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3361 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3421 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3481 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3541 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3601 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3661 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3721 ttcctggcct tttgctggcc tttt
//
