LOCUS       sequence-488009-        7175 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Expresses Src mutant E524A (aka E527A) in Mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  TITLE     Ionizable networks mediate pH-dependent allostery in SH2 signaling 
            proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7175)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Flag-Src E524A
FEATURES             Location/Qualifiers
     source          1..7175
                     /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"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1337..2131)
                     /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(1448..1467)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2058..2077
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2198..2527)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2212..2347
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2266..2285)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2512..2532
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2541..2969)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2651..2672)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2863..2882
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3015..3239
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3228..3245
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3268..3285)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3295..3336)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3362..3385)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3389..3412)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3423..3447
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5074..5098)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5127..5146)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5128..5146)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5191..5394)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5220..5240)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5395..5774
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5946..5965
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6040..6144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6145..6213,6214..7005)
                     /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     complement(6363..6382)
                     /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 agcgccacgc 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 gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgcg
     1321 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     1381 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     1441 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     1501 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     1561 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     1621 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     1681 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     1741 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     1801 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     1861 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     1921 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     1981 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     2041 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     2101 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     2161 cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa
     2221 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
     2281 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     2341 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     2401 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     2461 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     2521 gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2581 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2641 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2701 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2761 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2821 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2881 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2941 cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct
     3001 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     3061 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     3121 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     3181 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     3241 cgaggctgat cagcgggttt aaactcaatg gtgatggtga tgatgaccgg tacgcgtaga
     3301 atcgagaccg aggagagggt tagggatagg cttaccttcg aaccgcgggc cctctagatc
     3361 acttatcgtc gtcatccttg taatctccct tatcgtcgtc atccttgtaa tcctctagat
     3421 caaccacttt gtacaagaaa gctgggtcga ggttctcccc gggctggtac tggggcgcgg
     3481 tggacgtgaa gtagtcctcc aggaaggcct gcaggtactc gaaggtgggc cgctcctcag
     3541 gctccttccg ccagcactgg cacatgaggt cgtgcaggga ctcgggacac tccggcgggc
     3601 agggcatccg gtagccccgc tccacctggt ccagcacctc gcggttcacc atcccagggt
     3661 agggcacccg tccctttgtg gtgagctcag tcagcaggat cccgaaggac cacacgtccg
     3721 acttgatggt gaagcggcca tagagggcag cttctggagc cgtccacttg atggggaatt
     3781 tggcaccttg ccgcgccgtg tactcattgt cttcaatgag ccgagccagc ccaaagtcgg
     3841 ccactttgca caccaggttc tctcccacca ggatgttggc tgcacgaagg tcccggtgga
     3901 cgtagttcat ccgctccacg tacgccatgc ctgaggcgat ctgagcagcc atgtccacca
     3961 gctgaggcag ccgcaggtac ttgcctgtct cccccttgag aaagtccagc aaactcccct
     4021 tgctcatgta ctccgtgacg atgtaaatgg gctcctctga aaccacagca tacaactgca
     4081 ccagcttctc atgcctcagc ttcttcatga cctgggcctc ctgcaggaag gcctctggag
     4141 acatcgtgcc aggcttcagg gttttgatgg ccaccctggt ggtaccgttc caggtcccca
     4201 tccacacctc gccaaagcag ccctggccca gcttgacctc cagccgcagc gactcccgag
     4261 ggatctccca ggcatccttg gccaggccct gagtctgcgg cttggacgtg gggcacacgg
     4321 tggtgaggcg gtggcacagg ccatcggcgt gtttggagta gtaggccacc agctgctgca
     4381 ggctgttgaa ctgggtgcgg gaggtgatgt agaagccgcc gctgtccagc ttgcggatct
     4441 tgtagtgctt cacgttgagg cccttggcgt tgtcgaagtc agacactgag aggcagtagg
     4501 cacctttcgt ggtctcactt tctcgcacga ggaaggtccc tctcgggttc tctgcattga
     4561 gcagtaaccg ctctgactcc cgtctggtga tcttgccaaa ataccactcc tcagcctgga
     4621 tggagtcgga gggcgccacg tagttgctgg ggatgtagcc tgtctgtcct gtgctgagcg
     4681 agtgggccag ccaccagtct ccctctgtgt tgttgacaat ctggagccgc tcgcctttct
     4741 tgaaggacag gtctgtctcc gtcctagact catagtcata gagggccaca aaggtggtca
     4801 ctccaccggc cagcgggccc gccctctgcg gggaggtgac ggtgtccgag gagttgaagc
     4861 ctccgaacag cttgggctcg gcggccgcgg gggcgaaggc cgcgctgggg ccgcggtggc
     4921 cgtcggccga ggctggcttg ctgggggtct gcgaggcggg gaaagcgccc ccgccagcgc
     4981 cgtgcacgtt ctcggcgggc tccaggctgc ggcgccgctg gctggcatcc ttgggcttgc
     5041 tcttgttgct acccatggtt ctatctcctt cgaagcctgc ttttttgtac aaacttgttg
     5101 atagcttaac tagccagctt gggtctccct atagtgagtc gtattaattt cgataagcca
     5161 gtaagcagtg ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac
     5221 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     5281 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     5341 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     5401 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     5461 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     5521 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     5581 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     5641 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     5701 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     5761 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     5821 aaccctaagc agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat
     5881 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     5941 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc
     6001 gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6061 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6121 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6181 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6241 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6301 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6361 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6421 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6481 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6541 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6721 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6781 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6841 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7021 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7081 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7141 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
