LOCUS       sequence-74303-e        8266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vivo GFP based system to measure break induced replication (BIR) 
            repair efficiency. expression of I-SceI generates a double strand 
            break that when repaired by BIR mechanism will restore GFP. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8266)
  TITLE     pBIR-GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8266)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pBIR-GFP
FEATURES             Location/Qualifiers
     source          1..8266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1755..1775
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1797..1817
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2031..2040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2037..2039,2040..2042,2043..2555)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(2082..2103)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3259..3858)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3342..3362)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3839..3858
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3877..4374)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3925..3944)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4316..4339
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     complement(4462..4481)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4527..4582
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4581..4600)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    4965..5046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5214..5235)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5769..5785)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5769..5785)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5782..5804)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5793..5809
                     /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(5817..5823,5824..5841,5842..5847))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5862..5883
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5942..6137
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5988..6123
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6143..6277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6345..6362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6516..7104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6596..6615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7275..8066,8067..8135))
                     /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     7898..7917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8136..8240)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcagatcc gccgccacta
     1741 tgggatcaag atcgccaaaa aagaagagaa aggtgccgaa gaagcatgca gcaccaccaa
     1801 aaaaaaaacg aaaagtagaa gacccacgag gcaacaccag cggcgtgctg agcaccccca
     1861 aggccaagag ggccaagcac ccccccggca ccgagaagcc caggagcagg agccagagcg
     1921 agcagcccgc cacctgcccc atctgctacg ccgtgatcag gcagagcagg aacctgagga
     1981 ggcacctgga gctgaggcac ttcgccaagc ccggcgtgga tccaccggtc gccaccatgg
     2041 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2101 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2161 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2221 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2281 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2341 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2401 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2461 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2521 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgcctagg
     2581 gataacaggg taatcttaag gcgtaaattg taagcgttaa tctgaacctt ctgatggaat
     2641 tagaacttgg caaaacaata ctgagaatga agtgtatgtg gaacagaggc tgctgatctc
     2701 gttcttcagg ctatgaaact gacacatttg gaaaccacag tacttagaac cacaaagtgg
     2761 gaatcaagag aaaaacaatg atcccacgag agatctatag atctatagat catgagtggg
     2821 aggaatgagc tggcccttaa tttggttttg cttgtttaaa ttatgatatc caactatgaa
     2881 acattatcat aaagcaatag taaagagcct tcagtaaaga gcaggcattt atctaatccc
     2941 accccacccc cacccccgta gctccaatcc ttccattcaa aatgtaggta ctctgttctc
     3001 acccttctta acaaagtatg acaggaaaaa cttccatttt agtggacatc tttattgttt
     3061 aatagatcat caatttcgat ccgctcctgg gcaccgaact gcgccgcgtg ttcagcaggg
     3121 tcggcgtgtt cggtgtgtcc cccgcggtgg gcctcggggg cgggtgcggg gtcggcgggg
     3181 ccgccccggg tggcttcggt cggagccatg gggtcgtgcg ctcctttcgg tcgggcgctg
     3241 cgggtcgtgg ggcgggcgtc aggcaccggg cttgcgggtc atgcaccagg tgcgcggtcc
     3301 ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga aggggaggtt
     3361 gcggggcgcg gaggtctcca ggaaggcggg caccccggcg cgctcggccg cctccactcc
     3421 ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaga cgccgacggt
     3481 ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg
     3541 ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct cggcgaacac
     3601 cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc cgtcgtccgc
     3661 gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt gcagctcggt
     3721 gacccgctcg atgtggcggt ccgggtcgac ggtgtggcgc gtggcggggt agtcggcgaa
     3781 cgcggcggcg agggtgcgta cggcccgggg gacgtcgtcg cgggtggcga ggcgcaccgt
     3841 gggcttgtac tcggtcatgg tggcggctgg atcggtcgaa aggcccggag atgaggaaga
     3901 ggagaacagc gcggcagacg tgcgcttttg aagcgtgcag aatgccgggc ctccggagga
     3961 ccttcgggcg cccgccccgc ccctgagccc gcccctgagc ccgcccccgg acccacccct
     4021 tcccagcctc tgagcccaga aagcgaagga gcaaagctgc tattggccgc tgccccaaag
     4081 gcctacccgc ttccattgct cagcggtgct gtccatctgc acgagactag tgagacgtgc
     4141 tacttccatt tgtcacgtcc tgcacgacgc gagctgcggg gcggggggga acttcctgac
     4201 taggggagga gtagaaggtg gcgcgaaggg gccaccaaag aacggagccg gttggcgcct
     4261 accggtggat gtggaatgtg tgaggccaga ggccacttgt gtagcgccaa gtgcccagcg
     4321 gggctgctaa agcgcatgct ccagactgcc ttgggaaaag cgcctcccct acccggtaga
     4381 attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     4441 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     4501 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     4561 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     4621 tcagaatgag tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac
     4681 aaaggtggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     4741 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     4801 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     4861 cctgactact cccagtcata gctgtccctc ttctcttatg aagatccctc gacctgcagc
     4921 ccaagcttat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4981 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     5041 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     5101 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tctagagtcg
     5161 cggccgcttt acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     5221 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg
     5281 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     5341 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     5401 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     5461 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     5521 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     5581 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     5641 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     5701 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa aagcttggcg
     5761 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5821 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5881 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     5941 cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     6001 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     6061 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     6121 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     6181 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     6241 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     6301 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6361 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6421 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6481 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6541 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6601 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6661 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca aagctcacgc
     6721 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6781 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6841 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6901 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6961 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7021 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7081 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7141 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7201 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7261 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7321 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7381 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7441 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7501 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7561 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7621 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7681 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7741 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7801 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7861 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7921 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7981 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8041 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8101 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
