LOCUS       sequence-115389-        7971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses kinase dead AXL in mammalian cells with cistronic EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7971)
  AUTHORS   Meyer AS, Zweemer AJ, Lauffenburger DA
  TITLE     The AXL Receptor is a Sensor of Ligand Spatial Heterogeneity.
  JOURNAL   Cell Syst. 2015 Jul 29;1(1):25-36.
  PUBMED    26236777
REFERENCE   2  (bases 1 to 7971)
  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: "Cell 
            Syst."; date: "2015-07-29"; volume: "1(1)"; pages: "25-36"
COMMENT     Sequence Label: IRES-GFP-AXL-KD
FEATURES             Location/Qualifiers
     source          1..7971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3296..3319
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3330..3904,3905..3907,3908..3916)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3497..3514)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3724..3743
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3917..3919,3920..3922,3923..4636)
                     /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(3917..3919,3920..3922,3923..4633)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4223..4242)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4570..4591
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4759..4880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4796..4815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4850..4869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4887..5342)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5024..5045)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5236..5255
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5369..5473
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5475..5832
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5475..5666
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5498..5518)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5683..5818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5867..6661
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5867..6661
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5921..5940)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6531..6550
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6849..6868)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6893..6940
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7269..7857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7758..7777
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcgccacc atggcgtggc ggtgccccag
      661 gatgggcagg gtcccgctgg cctggtgctt ggcgctgtgc ggctgggcgt gcatggcccc
      721 caggggcacg caggctgaag aaagtccctt cgtgggcaac ccagggaata tcacaggtgc
      781 ccggggactc acgggcaccc ttcggtgtca gctccaggtt cagggagagc cccccgaggt
      841 acattggctt cgggatggac agatcctgga gctcgcggac agcacccaga cccaggtgcc
      901 cctgggtgag gatgaacagg atgactggat agtggtcagc cagctcagaa tcacctccct
      961 gcagctttcc gacacgggac agtaccagtg tttggtgttt ctgggacatc agaccttcgt
     1021 gtcccagcct ggctatgttg ggctggaggg cttgccttac ttcctggagg agcccgaaga
     1081 caggactgtg gccgccaaca cccccttcaa cctgagctgc caagctcagg gacccccaga
     1141 gcccgtggac ctactctggc tccaggatgc tgtccccctg gccacggctc caggtcacgg
     1201 cccccagcgc agcctgcatg ttccagggct gaacaagaca tcctctttct cctgcgaagc
     1261 ccataacgcc aagggggtca ccacatcccg cacagccacc atcacagtgc tcccccagca
     1321 gccccgtaac ctccacctgg tctcccgcca acccacggag ctggaggtgg cttggactcc
     1381 aggcctgagc ggcatctacc ccctgaccca ctgcaccctg caggctgtgc tgtcagacga
     1441 tgggatgggc atccaggcgg gagaaccaga ccccccagag gagcccctca cctcgcaagc
     1501 atccgtgccc ccccatcagc ttcggctagg cagcctccat cctcacaccc cttatcacat
     1561 ccgcgtggca tgcaccagca gccagggccc ctcatcctgg acccactggc ttcctgtgga
     1621 gacgccggag ggagtgcccc tgggcccccc tgagaacatt agtgctacgc ggaatgggag
     1681 ccaggccttc gtgcattggc aagagccccg ggcgcccctg cagggtaccc tgttagggta
     1741 ccggctggcg tatcaaggcc aggacacccc agaggtgcta atggacatag ggctaaggca
     1801 agaggtgacc ctggagctgc agggggacgg gtctgtgtcc aatctgacag tgtgtgtggc
     1861 agcctacact gctgctgggg atggaccctg gagcctccca gtacccctgg aggcctggcg
     1921 cccagtgaag gaaccttcaa ctcctgcctt ctcgtggccc tggtggtatg tactgctagg
     1981 agcagtcgtg gccgctgcct gtgtcctcat cttggctctc ttccttgtcc accggcgaaa
     2041 gaaggagacc cgttatggag aagtgtttga accaacagtg gaaagaggtg aactggtagt
     2101 caggtaccgc gtgcgcaagt cctacagtcg tcggaccact gaagctacct tgaacagcct
     2161 gggcatcagt gaagagctga aggagaagct gcgggatgtg atggtggacc ggcacaaggt
     2221 ggccctgggg aagactctgg gagagggaga gtttggagct gtgatggaag gccagctcaa
     2281 ccaggacgac tccatcctca aggtggctgt gaggacgatg aagattgcca tctgcacgag
     2341 gtcagagctg gaggatttcc tgagtgaagc ggtctgcatg aaggaatttg accatcccaa
     2401 cgtcatgagg ctcatcggtg tctgtttcca gggttctgaa cgagagagct tcccagcacc
     2461 tgtggtcatc ttacctttca tgaaacatgg agacctacac agcttcctcc tctattcccg
     2521 gctcggggac cagccagtgt acctgcccac tcagatgcta gtgaagttca tggcagacat
     2581 cgccagtggc atggagtatc tgagtaccaa gagattcata caccgggacc tggcggccag
     2641 gaactgcatg ctgaatgaga acatgtccgt gtgtgtggcg gacttcgggc tctccaagaa
     2701 gatctacaat ggggactact accgccaggg acgtatcgcc aagatgccag tcaagtggat
     2761 tgccattgag agtctagctg accgtgtcta caccagcaag agcgatgtgt ggtccttcgg
     2821 ggtgacaatg tgggagattg ccacaagagg ccaaacccca tatccgggcg tggagaacag
     2881 cgagatttat gactatctgc gccggggaaa tcgcctgaag cagcctgcgg actgtctgga
     2941 tggactgtat gccttgatgt cgcggtgctg ggagctaaat ccccaggacc ggccaagttt
     3001 tacagagctg cgggaagatt tggagaacac actgaaggcc ttgcctcctg cccaggagcc
     3061 tgacgaaatc ctctatgtca acatggatga gggtggaggt tatcctgaac cccctggagc
     3121 tgcaggagga gctgaccccc caacccagcc agaccctaag gattcctgta gctgcctcac
     3181 tgcggctgag gtccatcctg ctggacgcta tgtcctctgc ccttccacaa cccctagccc
     3241 cgctcagcct gctgataggg gctccccagc agccccaggg caggaggatg gtgccgacta
     3301 caaagacgat gacgacaagt agggatccgc ccctctccct cccccccccc taacgttact
     3361 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     3421 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     3481 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     3541 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     3601 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     3661 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     3721 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     3781 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta
     3841 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     3901 taatatggcc acaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3961 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     4021 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     4081 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     4141 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     4201 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     4261 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     4321 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4381 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4441 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4501 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4561 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4621 cgagctgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac cacatttgta
     4681 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     4741 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4861 aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     4921 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     4981 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5041 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     5101 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     5161 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     5221 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5281 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5341 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5401 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5461 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     5521 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     5581 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     5641 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     5701 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5761 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5821 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     5881 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     5941 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     6001 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     6061 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     6121 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     6181 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     6241 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     6301 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     6361 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     6421 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     6481 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     6541 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     6601 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     6661 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     6721 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     6781 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     6841 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     6901 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     6961 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     7021 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     7081 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     7141 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7201 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7261 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7321 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7381 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7441 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7501 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7561 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7621 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7681 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7741 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7801 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7861 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7921 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
