LOCUS       sequence-491434-        6930 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  Encodes GFP downstream of an attP/attB flanked Neomycin stop 
            cassette driven by the CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6930)
  AUTHORS   Schick E, McCaffery SD, Keblish EE, Thakurdin C, Emerson MM
  TITLE     Lineage tracing analysis of cone photoreceptor associated 
            cis-regulatory elements in the developing chicken retina.
  JOURNAL   Sci Rep. 2019 Jun 27;9(1):9358. doi: 10.1038/s41598-019-45750-7.
  PUBMED    31249345
REFERENCE   2  (bases 1 to 6930)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2019-06-27"; volume: "9(1):9358. doi"; pages: " 
            10.1038/s41598-019-45750-7"
COMMENT     Sequence Label: CAaNa::GFP
FEATURES             Location/Qualifiers
     source          1..6930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          675..1691
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1615..1637
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1699..1718
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1766..1799)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1896..2690
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1950..1969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2560..2579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2870..3004
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3019..3088
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     regulatory      3160..3169
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3166..3168,3169..3171,3172..3885)
                     /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(3166..3168,3169..3171,3172..3882)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3211..3232)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3472..3491)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3819..3840
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3972..3991)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4037..4092
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4091..4110)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4453..4469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4453..4469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4466..4488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4477..4493
                     /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(4501..4507,4508..4525,4526..4531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4546..4567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4652..4786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4806..4940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5008..5025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5179..5767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5259..5278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5938..6729,6730..6798))
                     /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     6561..6580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6799..6903)
                     /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 agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggggggggg ggggggggcg cgcgccaggc ggggcggggc
      541 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
      601 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
      661 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
      721 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      781 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
      841 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
      901 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
      961 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1021 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1081 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1141 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1201 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1261 tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc
     1321 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1381 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1441 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1501 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1561 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     1621 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     1681 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     1741 aattgattaa ttcgagcgaa cgcgtataac ttcgtatagc atacattata cgaagttatc
     1801 tcgaggtgcc ccaactgggg taacctttga gttctctcag ttgggggcgt agtcggattt
     1861 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     1921 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     1981 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     2041 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     2101 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     2161 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     2221 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     2281 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     2341 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     2401 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     2461 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     2521 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     2581 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     2641 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     2701 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     2761 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     2821 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccatcgata acttgtttat
     2881 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2941 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3001 gatcaaatcc gaacgcgtct cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc
     3061 cccgggcgcg tactccacct cacccatcct cgagtcgctc ggtacgattt aaattgaatt
     3121 ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagcaagggc
     3181 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3241 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3301 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3361 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3421 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3481 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3541 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3601 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3661 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3721 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3781 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3841 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcactcct
     3901 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     3961 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4021 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4081 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4141 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     4201 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     4261 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4321 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4381 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt
     4441 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4501 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4561 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     4621 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc
     4681 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4741 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4801 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4861 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4921 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4981 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5041 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5101 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5161 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5221 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5281 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5341 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5401 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5461 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5521 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5581 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5641 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5761 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5821 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5881 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5941 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6001 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6061 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6121 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6181 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6241 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6301 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6361 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6421 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6481 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6541 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6601 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6661 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6721 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6781 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6841 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6901 gcgcacattt ccccgaaaag tgccacctgg
//
