LOCUS       sequence-434871-        6254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SUMO tagged human gamma A crystallin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6254)
  TITLE     Completion of previous SUMO tagged human crystallin submission plus 
            plasmids for native chemical ligation to produced gamma S crystallin
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6254)
  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            "Unpublished"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: PE-SUMO CRYGA
FEATURES             Location/Qualifiers
     source          1..6254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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)."
     RBS             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             144..431
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             1006..1023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1076..1094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1090..1137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1174..1629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1261..1280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1471..1492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1655..1759
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1760..1828,1829..2620)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1978..1997)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2791..3379
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3280..3299
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3533..3550
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3565..3704
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3630..3649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3790..3812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3806..3997)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4806..5888)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5850..5869
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5889..5966)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6172..6191
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc
      121 atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca
      181 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
      241 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
      301 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
      361 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac
      421 agattggagg tgggaagatc accttctacg aggaccgaga ctttcagggt cgctgctaca
      481 attgcatcag tgactgcccc aacctgcggg tctacttcag ccgctgcaac tccatccgag
      541 tagacagcgg ctgctggatg ctctatgagc gtcccaatta ccagggccac cagtacttcc
      601 tgcgccgagg caagtacccc gactatcagc actggatggg cctcagcgac tcggtccaat
      661 cctgccgtat aattcctcat accagctcgc acaagttaag gctgtacgag agagatgact
      721 accgaggcct tatgtctgag ctcactgatg actgcgcctg tgttccagaa ctgttccgtc
      781 tccctgagat ctattccctc cacgtactgg agggctgctg ggtcctctat gaaatgccca
      841 actaccgggg gcggcagtat ctgctgaggc ctggggacta cagaaggtac cacgactggg
      901 ggggtgcaga tgccaaagtc ggctctttga gacgggtcac cgatttgtac taatctagag
      961 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     1021 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1081 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1141 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1621 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1681 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1741 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1801 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1861 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1921 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1981 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     2041 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2101 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2161 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2221 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     2281 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2341 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2401 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2461 agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2521 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2581 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2641 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2701 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2761 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2821 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2881 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2941 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3001 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3061 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3121 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3181 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3241 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3301 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3361 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3421 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3481 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3541 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3601 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3661 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3721 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3781 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3841 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3901 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3961 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4021 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4081 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4141 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4201 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4261 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4321 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4381 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4441 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     4501 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4561 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4621 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4681 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4741 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4921 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4981 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5041 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5101 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5161 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5221 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5281 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     5341 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     5401 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     5461 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     5521 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     5581 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5641 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5701 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5761 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5821 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5881 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5941 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     6001 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     6061 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     6121 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     6181 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     6241 ggcgtagagg atcg
//
