LOCUS       sequence-464635-        6755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control retroviral vector expressing Green Fluorescent Protein. 
            Puromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6755)
  AUTHORS   Addgene Research Program
  TITLE     Addgene Retroviral Backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6755)
  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            "Unpublished"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: pAG Retro EGFP Puro
FEATURES             Location/Qualifiers
     source          1..6755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     50..66
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     51..67
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             92..561
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    626..825
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1026..1442
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1372..1394
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1410..1426
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             join(1453..1455,1456..1458,1459..2172)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1453..1455,1456..1458,1459..2169)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1498..1519)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1759..1778)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2106..2127
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2209..2229)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2214..2543
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2394..2529
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2553..3152
                     /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(2553..2572)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3049..3069
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3373..3842
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3948..3968)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3948..3966)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3987..4003)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3987..4003)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4000..4022)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4011..4027
                     /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(4035..4041,4042..4059,4060..4065))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4080..4101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4218..4235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4389..4977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4469..4488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5148..5939,5940..6008))
                     /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     5771..5790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6009..6113)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6139..6594)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6276..6297)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6488..6507
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6721..6743
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6735..6752
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6736..6752
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac
       61 ggtatcgggg gcatgcagtg ccacctgcag cctgaatatg ggccaaacag gatatctgtg
      121 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca
      181 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc
      241 cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc
      301 aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc
      361 tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc
      421 gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca
      481 gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
      541 cccgtcagcg ggggtctttc atttgggggc tcgtccggga tcgggagacc cctgcccagg
      601 gaccaccgac ccaccaccgg gaggcaagct ggccagcaac ttatctgtgt ctgtccgatt
      661 gtctagtgtc tatgactgat tttatgcgcc tgcgtcggta ctagttagct aactagctct
      721 gtatctggcg gacccgtggt ggaactgacg agttctgaac acccggccgc aaccctggga
      781 gacgtcccag ggactttggg ggccgttttt gtggcccgac ctgaggaagg gagtcgatgt
      841 ggaatccgac cccgtcagga tatgtggttc tggtaggaga cgagaaccta aaacagttcc
      901 cgcctccgtc tgaatttttg ctttcggttt ggaaccgaag ccgcgcgtct tgtctgctgc
      961 agcgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa
     1021 attagggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg
     1081 agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct
     1141 ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc
     1201 tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg
     1261 tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct
     1321 ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac
     1381 ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg
     1441 ccggccggat ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1501 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1561 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1621 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1681 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1741 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1801 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1861 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1921 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1981 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2041 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     2101 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2161 ctgtacaagt aatacgtagg aattcgccag cacagtggtc gaccctgtgg aatgtgtgtc
     2221 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2281 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2341 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2401 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2461 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     2521 ttggaggcct aggcttttgc aaaaagctta ccatgaccga gtacaagccc acggtgcgcc
     2581 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     2641 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     2701 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     2761 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     2821 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     2881 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     2941 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3001 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     3061 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3121 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     3181 ccgaaaggag cgcacgaccc catgcatcga taaaataaaa gattttattt agtctccaga
     3241 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
     3301 tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa
     3361 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     3421 gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa
     3481 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc
     3541 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     3601 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     3661 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact
     3721 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
     3781 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
     3841 cacatgcagc atgtatcggc gccaagccga attctgcaga tatccatcac actggcggcc
     3901 gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     3961 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4021 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4081 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4141 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4201 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4261 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4321 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4381 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4441 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4501 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4561 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4621 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4681 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4741 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4801 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4861 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4921 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4981 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5041 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5101 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5161 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5221 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5281 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5341 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5401 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5461 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5521 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5581 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5641 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5701 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5761 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5821 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5881 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5941 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6001 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6061 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6121 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6181 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6241 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6301 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6361 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6421 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6481 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6541 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     6601 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     6661 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     6721 cccagtcacg acgttgtaaa acgacggcca gtgag
//
