LOCUS       sequence-464134-        5915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of spectrin beta chain, 
            non-erythrocytic 1 (Beta-II spectrin) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5915)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5915)
  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"
COMMENT     Sequence Label: pAIP4_1BKR
FEATURES             Location/Qualifiers
     source          1..5915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     716..735
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     800..819
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      930..1518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1419..1438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1672..1689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1704..1843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1769..1788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1929..1951
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1945..2136)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2945..4027)
                     /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     3989..4008
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4028..4105)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4311..4330
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4418..4437
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4418..4436
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4437..4461
                     /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             4476..4498
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4521..4550
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5193..5210
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5263..5281)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5277..5324
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5361..5816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5448..5467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5658..5679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5908..5915,1..808))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(5908..5915,1..808))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
       61 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
      121 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
      181 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
      241 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
      301 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
      361 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
      421 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
      481 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
      541 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
      601 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
      661 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
      721 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
      781 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
      841 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      901 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      961 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1021 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1081 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1141 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1201 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1261 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1321 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1381 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1441 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1501 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1561 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     1621 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     1681 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1741 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1801 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     1861 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     1921 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     1981 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     2041 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     2101 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     2161 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     2221 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     2281 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     2341 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     2401 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     2461 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     2521 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     2581 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     2641 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     2701 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     2761 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     2821 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     2881 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     2941 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3001 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3061 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3121 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     3181 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     3241 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3301 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     3361 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3421 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3481 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     3541 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     3601 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     3661 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     3721 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     3781 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     3841 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     3901 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     3961 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4021 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4081 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     4141 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     4201 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     4261 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     4321 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     4381 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     4441 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     4501 tacatatgat ggcgaagatt catcaccatc atcaccatca ccatcaccac ggtagcctgc
     4561 aagattcgga agtcaaccaa gaagcaaagc cggaagtcaa gccggaagtg aaaccggaaa
     4621 cccatattaa cctgaaagtt agtgacggca gctctgaaat tttctttaag atcaaaaaga
     4681 ccacgccgct gcgtcgcctg atggaagcgt ttgccaaacg tcagggcaag gaaatggata
     4741 gcctgcgttt cctgtatgac ggtattcgca tccaggcaga tcaagcgccg gaagacctgg
     4801 acatggaaga caacgacatc attgaagccc accgtgaaca gattggcggc tctggaaagt
     4861 cagctaaaga tgcactattg ttatggtgcc agatgaaaac tgcgggttat ccaaatgtta
     4921 atattcacaa ctttaccacc agctggcgcg acggcatggc gtttaacgcg ctgatccaca
     4981 aacatcgtcc ggacctgatt gatttcgaca agctgaaaaa gtccaacgcc cactacaatc
     5041 tccaaaacgc tttcaacctg gcagagcagc atctgggttt gaccaagttg ctggatccgg
     5101 aagatattag cgttgatcat ccggacgaga agtctatcat cacctacgtg gtgacgtact
     5161 atcactattt cagcggttcg taatgactcg agcaccacca ccaccaccac tgagatccgg
     5221 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     5281 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     5341 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5401 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5461 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5521 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5581 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5641 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5701 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5761 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     5821 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5881 aatatgtatc cgctcatgaa ttaattctta gaaaa
//
