LOCUS       sequence-494249-        5635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning backbone for cloning bridge RNAs for ISCro4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5635)
  AUTHORS   Perry NT, Bartie LJ, Katrekar D, Gonzalez GA, Durrant MG, Pai JJ, 
            Fanton A, Martins JQ, Hiraizumi M, Ricci-Tam C, Nishimasu H, 
            Konermann S, Hsu PD
  TITLE     Megabase-scale human genome rearrangement with programmable bridge 
            recombinases.
  JOURNAL   Science. 2025 Sep 25:eadz0276. doi: 10.1126/science.adz0276.
  PUBMED    40997214
REFERENCE   2  (bases 1 to 5635)
  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: "Science. 
            2025 Sep 25:eadz0276. doi: 10.1126/science.adz0276."
COMMENT     Sequence Label: pNP2922 [pNP2321 - Cloning backbone, bRNA Land...
FEATURES             Location/Qualifiers
     source          1..5635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /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(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1937..2185
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1937..1957
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2108..2127
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           2221..2288
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     promoter        2355..3533
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2586..3524
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3481..3501
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3540..3549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4345..4354
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4545..4565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4566..4586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4587..4607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4617..4673
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(4680..4682,4683..5396)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4680..4682,4683..5393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4722..4743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4983..5002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5330..5351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5421..5438)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5427..5634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt gggtcacgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1921 gagcgaggaa gcggaagagg gcctatttcc catgattcct tcatatttgc atatacgata
     1981 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2041 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2101 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2161 tatcttgtgg aaaggacgaa acaccgggag acgatggcca cccgtctcct tttatttttt
     2221 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacatgctt cggcatggcg
     2281 aatgggactt tttttgttct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg
     2341 gctctagagg taccggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2401 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2461 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2521 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2581 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
     2641 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
     2701 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
     2761 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
     2821 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
     2881 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
     2941 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
     3001 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
     3061 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
     3121 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     3181 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     3241 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     3301 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     3361 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     3421 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     3481 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg
     3541 ccaccatgga acaggagctg cacttcatcg gcatcgacgt gtccaaagca aagctggacg
     3601 tggacgtgct gcgacctgac ggcagacaca gaagcaagaa attcgctaat acccctaagg
     3661 gacacgacga gctgctgaga tggctgagcg gccacagagt ggccccagct cacatctgca
     3721 tggaagctac aagcacctac atggaggatg tggccgccca cctgagcgac gccggctaca
     3781 ccgtgtccgt gatcaaccct gccctgggca aggcctttgc ccaaagcgag ggactgcgaa
     3841 gcaagacaga tgctgttgat gccagaatgc tggccgagtt ctgtcggcag aagcggcctc
     3901 cagcctggga ggcccctcac cctgtggaac gggccctgag ggccctcgtg ctgcgccacc
     3961 agagcctgac cgatatgcac acccaggagc tgaaccgact ggaaaccgcc cgggaagtgc
     4021 agcggcctag cattgacgcc catctgctgt ggctgcacgc cgagctgaaa agaatcgaga
     4081 aacagatcaa ggacctgaca gatgatgacc ccgacatgaa gcaccggcgg aagctgctgg
     4141 aaagcatccc cggcatcgga gaaaagacca gcgccgtcct gctcgcttat acaggcctga
     4201 aggaaagatt cacccacgcc agacagttcg ctgcttttgc cggcctgaca cctagaagat
     4261 acgagagcgg cagctctgtg aaccgggcct ctagaatgag caaggccgga catgcctctc
     4321 tgcggagagc cctgtacatg cctgccatgg tggccgtgtc taagaccgag tggggcagag
     4381 ccttccggga cagactggcc ggcaacggca agaagggcaa agtgatcatc ggcgccatga
     4441 tgagaaagct ggcccaggtg gcctacggcg tgctgaagtc cggcgtcccc ttcgacgcca
     4501 gcagacacaa ccccgtggct gctgctagcg gagggggtgg aagcccaaaa aagaagagaa
     4561 aggtacctaa gaaaaagcgg aaggtcccca agaagaagag gaaagttgga agcggtgcca
     4621 cgaacttctc tctgttaaag caagcaggcg acgtagaaga aaacccaggt cccggatccg
     4681 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     4741 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     4801 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     4861 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     4921 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     4981 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     5041 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     5101 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     5161 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     5221 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     5281 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     5341 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaactcg
     5401 agctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5461 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5521 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5581 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggac
//
