LOCUS       sequence-247197-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin resistance gene and iCre recombinase (version 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     2A plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6473)
  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            "Unpublished"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: pCAGGS-pac-T2A-iCre (v2)
FEATURES             Location/Qualifiers
     source          1..6473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        11..390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        392..669
                     /label=chicken beta-actin promoter
     intron          670..1686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1610..1632
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1744..2340
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MAEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2240..2260
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2362..2415
                     /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             2437..2457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3501..3520)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3566..3621
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3620..3639)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3982..3998)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3982..3998)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3995..4017)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4006..4022
                     /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(4030..4036,4037..4054,4055..4060))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4075..4096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4155..4350
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4201..4336
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4356..4490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4393..4412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4447..4466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4558..4575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4729..5317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4809..4828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5488..6279,6280..6348))
                     /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     6111..6130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6349..6453)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgggtc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
       61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      361 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
      421 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
      481 tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc
      541 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      601 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
      661 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
      721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      841 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      901 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     1021 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     1081 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1201 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     1321 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1381 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     1441 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     1501 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     1561 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     1621 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1741 gccatggccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     1801 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     1861 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     1921 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     1981 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     2041 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     2101 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     2161 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     2221 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     2281 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     2341 ggcggctccg gcggccgctc cgagggccgc ggctccctgc tgacctgcgg cgacgtggag
     2401 gagaaccccg gccccgcccc cggctccacc atggtgccca agaagaagag gaaagtctcc
     2461 aacctgctga ctgtgcacca aaacctgcct gccctccctg tggatgccac ctctgatgaa
     2521 gtcaggaaga acctgatgga catgttcagg gacaggcagg ccttctctga acacacctgg
     2581 aagatgctcc tgtctgtgtg cagatcctgg gctgcctggt gcaagctgaa caacaggaaa
     2641 tggttccctg ctgaacctga ggatgtgagg gactacctcc tgtacctgca agccagaggc
     2701 ctggctgtga agaccatcca acagcacctg ggccagctca acatgctgca caggagatct
     2761 ggcctgcctc gcccttctga ctccaatgct gtgtccctgg tgatgaggag aatcagaaag
     2821 gagaatgtgg atgctgggga gagagccaag caggccctgg cctttgaacg cactgacttt
     2881 gaccaagtca gatccctgat ggagaactct gacagatgcc aggacatcag gaacctggcc
     2941 ttcctgggca ttgcctacaa caccctgctg cgcattgccg aaattgccag aatcagagtg
     3001 aaggacatct cccgcaccga tggtgggaga atgctgatcc acattggcag gaccaagacc
     3061 ctggtgtcca cagctggtgt ggagaaggcc ctgtccctgg gggttaccaa gctggtggag
     3121 agatggatct ctgtgtctgg tgtggctgat gaccccaaca actacctgtt ctgccgggtc
     3181 agaaagaatg gtgtggctgc cccttctgcc acctcccaac tgtccacccg ggccctggaa
     3241 gggatctttg aggccaccca ccgcctgatc tatggtgcca aggatgactc tgggcagaga
     3301 tacctggcct ggtctggcca ctctgccaga gtgggtgctg ccagggacat ggccagggct
     3361 ggtgtgtcca tccctgaaat catgcaggct ggtggctgga ccaatgtgaa cattgtgatg
     3421 aactacatca gaaacctgga ctctgagact ggggccatgg tgaggctgct cgaggatggg
     3481 gactgataag gcgcgccgga tctttttccc tctgccaaaa attatgggga catcatgaag
     3541 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     3601 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     3661 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     3721 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     3781 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     3841 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     3901 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     3961 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4021 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4081 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4141 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4201 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4261 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4321 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     4381 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4441 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4801 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgc
//
