LOCUS       sequence-466029-        5755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin resistance/eGFP cassette in mammalian cells (transfection 
            marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5755)
  AUTHORS   Witte IP, Lampe GD, Eitzinger S, Miller SM, Berrios KN, McElroy AN, 
            King RT, Stringham OG, Gelsinger DR, Vo PLH, Chen AT, Tolar J, 
            Osborn MJ, Sternberg SH, Liu DR
  TITLE     Programmable gene insertion in human cells with a laboratory-evolved
            CRISPR-associated transposase.
  JOURNAL   Science. 2025 May 15;388(6748):eadt5199. doi: 
            10.1126/science.adt5199. Epub 2025 May 15.
  PUBMED    40373119
REFERENCE   2  (bases 1 to 5755)
  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."; 
            date: "2025-05-15"; volume: "388(6748):eadt5199. doi"; pages: " 
            10.1126/science.adt5199. Epub 2025 May 15"
COMMENT     Sequence Label: pCMV-PuroR-T2A-eGFP
FEATURES             Location/Qualifiers
     source          1..5755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1353..1560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1549..1566
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(1594..2304,2305..2307))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1594..2304,2305..2307))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1636..1657)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1985..2004
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2244..2265
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2308..2361)
                     /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             complement(2371..2967)
                     /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(2451..2471)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2948..2967
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3051..3254)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3080..3100)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3255..3634
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(3952..3971)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3953..3971)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3981..3998)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3981..3997)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3990..4012)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4138..4593)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4275..4296)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4487..4506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4620..4724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4725..4793,4794..5585)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4943..4962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgg ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggtaccgg gcccgtttaa actacagttg aagtcggaag
     1081 tttacataca ccttagccaa atacatttaa actcactttt tcacaattcc tgacatttaa
     1141 tccgagtaaa gattccctgt cttaaggtca gttaggatca ccactttatt ttaagaatgt
     1201 gaaatatcag aataatagta gagagaatga ttcatttcag cttttatttc tttcatcaca
     1261 ttcccagtgg gtcagaagtg tacatacacg cgcttagtat ttggtagcat tgcctttaaa
     1321 ttgtttaaag atctggccat ctagacagta agtccccagc atgcctgcta ttctcttccc
     1381 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
     1441 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
     1501 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag
     1561 tcgaggctga tcagcgagct ctagttagaa ttccttgtac agctcgtcca tgccgagagt
     1621 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1681 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1741 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     1801 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     1861 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     1921 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     1981 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     2041 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     2101 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     2161 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     2221 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     2281 ggtgaacagc tcctcgccct tgctcactgg gccaggattc tcctcgacgt caccgcatgt
     2341 tagcagactt cctctgccct ctccactgcc ggcaccgggc ttgcgggtca tgcaccaggt
     2401 gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa
     2461 ggggaggttg cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc
     2521 ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt cgggcgagac
     2581 tccgacggtg gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc
     2641 catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg ggccgatctc
     2701 ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc
     2761 gtcgtccgcg acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg
     2821 cagctcggtg acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg tggcggggta
     2881 gtcggcgaac gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc gggtggcgag
     2941 gcgcaccgtg ggcttgtact cggtcatggt ggctaccggt aagcttcgtg atgtccagtg
     3001 ctattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt
     3061 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     3121 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     3181 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     3241 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     3301 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     3361 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     3421 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     3481 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     3541 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     3601 gattactatt aataactagt caataatcaa tgtcaattaa ttcgcggccg cgcgcactag
     3661 tgaattccat ggatatcaag cttaaaggca cagtcaagcg cgtgtatgta cacttctgac
     3721 ccactggaat tgtgatacag tgaattataa gtgaaataat ctgtctgtaa acaattgttg
     3781 gaaaaatgac ttgtgtcatg cacaaagtag atgtcctaac tgacttgcca aaactattgt
     3841 ttgttaacaa gaaatttgtg gagtagttga aaaacgagtt ttaatgactc caacttaagt
     3901 gtatgtaaac ttccgacttc aactgtaggt ttaaacgcta gctccaattc gccctatagt
     3961 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4021 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4081 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa
     4141 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4201 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4261 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4321 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4381 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4441 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4501 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccactc
     4561 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     4621 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4681 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4741 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4801 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4861 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4921 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4981 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5041 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5101 ccatgagtga taacactgcg gccaagttac ttctgacaac gatcggagga ccgaaggagc
     5161 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5221 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5281 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5341 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5401 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5461 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5521 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5581 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5641 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5701 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
